CD44 polypeptides, polynucleotides encoding same, antibodies directed thereagainst and method of using same for diagnosing and treating inflammatory diseases

ABSTRACT

An isolated polypeptide is provided. The isolated polypeptide comprising an antigen recognition domain capable of specifically binding a CD44 polypeptide as set forth in SEQ ID NO: 2 and incapable of binding a CD44 polypeptide selected from the group consisting of: SEQ ID NO: 4 or 6.

This application is a divisional of U.S. patent application Ser. No.12/230,899, filed Sep. 8, 2008, now U.S. Pat. No. 7,883,857, which is acontinuation of U.S. patent application Ser. No. 11/130,206, filed May17, 2005, now U.S. Pat. No. 7,534,605 issued on May 19, 2009, which is aContinuation-In-Part of PCT Patent Application No. PCT/IL2004/000639,filed Jul. 15, 2004, which claims the benefit of priority from U.S.Provisional Patent Application No. 60/495,876, filed Aug. 19, 2003, andfrom U.S. Provisional Patent Application No. 60/486,919, filed Jul. 15,2003.

U.S. patent application Ser. No. 11/130,206 is also aContinuation-In-Part of U.S. patent application Ser. No. 10/012,969,filed Dec. 7, 2001, now abandoned, which is a Continuation-In-Part ofPCT Patent Application No. PCT/IL00/00326, filed Jun. 7, 2000, whichclaims the benefit of priority from Israel Patent Application No.133647, filed Dec. 21, 1999, and from Israel Patent Application No.130356, filed Jun, 8, 1999. The contents of the above applications areall incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to polypeptides of a novel CD44 variantand to polynucleotides encoding same. Specifically the present inventionrelates to oligonucleotides specific for the novel CD44 polynucleotidevariant, antibodies specific for the novel CD44 polypeptide variant, acell hybridoma expressing monoclonal antibodies (mAbs) specific for saidRA specific CD44 variant and mAb expressed thereby, and the use of theseantibodies and oligonucleotides in the diagnosis and treatment ofinflammatory diseases such as rheumatoid arthritis.

The following is a list of references which are intended for betterunderstanding of the background of the present invention:

Aune, T. M., et al., Published EP Application No. 501233 (1992).

Hale, L. P., et al., WO 9409811 (1994)/Herrlich

et al., European Patent No. 538754, (1991).

Jalkanen, S., et al., WO 9500658, (1993).

Koopman et al., J. Exp. Med. 177:897-904 (1993)

Naor, D., et al., Adv. Cancer Res., 71:241, (1997).

Screaton, G. R., et al., Proc. Natl. Acad. Sci. USA, 89:12160, (1992).

Verdrengh, M., et al., Scand J. Immunol., 42:353, (1995)

The cell surface adhesion molecule, designated CD44, has been shown tobe implicated in cell-cell and cell-matrix interactions, as well as incell traffic and cell transendothelial migration.

CD44 is a single chain molecule comprising a conserved amino terminalextracellular domain, a nonconserved membrane proximal region, avariable region expressing various combinations of variant exons, aconserved transmembrane spanning domain and a conserved cytoplasmictail. The genomic sequence of mouse and human CD44 includes 5 constantexons at the 5′ terminus, and 5 constant exons at the 3′ end. The mouseCD44 gene includes 10 variant exons in the middle of the molecule,designated V₁-V₁₀, resulting in a total of 20 exons. The human CD44 genecomprises only 9 of these 10 variant exons (V₂-V₁₀) thus comprising atotal of 19 exons (Screaton, G. R., et al., 1992). Differential V₂-V₁₀alternative splicing generates many isoforms of CD44 that expressvarious combinations of variant exons (designated exon Vx, x=1-10),which are inserted in the membrane proximal domain and constitute thevariable region of the molecule. These molecules are designated CD44variants (CD44v). To date, a few dozen isoforms of CD44 are known.

In standard CD44 (CD44s, SEQ ID NO:5), constant exon number 5 is spliceddirectly to constant exon number 16 and therefore this molecule lacksthe entire variable region. The resulting protein product is expressedpredominantly on hematopoietic cells and therefore, this product is alsoknown as hematopoietic CD44 (CD44H) or standard CD44 product (CD44sproduct, SEQ ID NO:6). In keratinocyte CD44, the longest CD44 identifiedso far, exon V3 to exon V10 are inserted in tandem between the twoconstant regions of the molecule.

The CD44 N-terminus contains the ligand binding site of the molecule.Hyaluronic acid (HA) is th ge principal ligand of CD44, but otherextracellular matrix (ECM) components (e.g. laminin, collagen,fibronectin and chondroitin sulfate) as well as non-ECM constituents(mucosal vascular addressin, serglycin, osteopontin and class IIinvariant chain) can also interact with the CD44 receptor. Markedaccumulation of CD44, and sometimes hyaluronic acid, is detected inareas of intensive cell migration and cell proliferation, as in woundhealing, tissue remodeling, inflammation (including auto inflammation),morphogenesis and carcinogenesis.

The involvement of CD44 protein and variants thereof in autoimmunediseases is known. For example, it has been shown that anti-CD44monoclonal antibodies (mAbs) can ameliorate the severity ofexperimentally induced autoimmune arthritis in mice (Verdrengh, M. etal. 1995). However, these mAbs are directed against the constant regionof CD44 (and are thus designated anti-pan CD44 mAbs shared by all CD44isoforms). Therefore, such mAbs may also block CD44 expressed on normalcells, which is required for migratory activity of immune andinflammatory cells engaged in microorganism eradication.

Monoclonal Abs directed against various variant regions of CD44 havealso been suggested as potential agents for treatment of autoimmunediseases. Herrlich et al. describe mAbs directed againstmetastasis-specific variants of CD44v surface protein of a ratpancreatic adenocarcinoma (Herrlich et al., 1991). Anti-CD44-monoclonalantibodies, which inhibit T-cell proliferation, were also provided fortreatment of various autoimmune diseases (Aune, T. M. et al., 1992).Monoclonal antibodies specific for forms of CD44 containing exon v6 werealso reported as being useful for diagnosing inflammatory diseases(Jalkanen, S. et al., 1994). In addition, it has been reported (Hale, L.P., 1992) that administration of a CD44 protein, peptide or derivativecan be used for treating various autoimmune diseases.

In an experimental arthritis mouse model (collagen-induced arthritis),injection of one of three different anti-CD44 mAbs, but not of theisotype-matched control mAbs, at disease onset, prevented an increase infootpad swelling and helped to maintain the clinical score at a very lowlevel (Nedvetzki et al. 1999). Each of the three different types ofanti-CD44 mAb recognized a distinct constant epitope of the CD44receptor. All three antibodies displayed a similar anti-arthriticeffect.

The involvement of CD44 in malignant processes has also been described(Naor, D., 1997). Anti-CD44 mAbs which were injected into mice, wereshown to inhibit or prevent infiltration of various lymphoma andcarcinoma cells into their target organs. In addition, transfection of avariant CD44 isoform into non metastatic rat pancreatic adenocarcinomacells conferred metastatic potential to these cells.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anisolated polypeptide comprising an antigen recognition domain capable ofspecifically binding a CD44 polypeptide as set forth in SEQ ID NO: 2 andincapable of binding a CD44 polypeptide selected from the groupconsisting of: SEQ ID NO: 4 or 6.

According to another aspect of the present invention there is providedan isolated polypeptide comprising a CD44 variant polypeptide whichcomprises contiguously a first amino acid sequence being at least 90%homologous to amino acid coordinates 1-302 of SEQ ID NO: 2, an alanineresidue and a second amino acid sequence being at least 90% homologousto amino acid coordinates 304-700 of SEQ ID NO:2.

According to yet another aspect of the present invention there isprovided an isolated polypeptide comprising an amino acid sequence asset forth in SEQ ID NO: 2.

According to still another aspect of the present invention there isprovided a peptide of at least 8 amino acids derived from the isolatedpolypeptide and which comprises the alanine residue.

According to an additional aspect of the present invention there isprovided an isolated polypeptide comprising an antigen recognitiondomain capable of specifically binding the isolated polypeptide of claim7 and incapable of binding a CD44 polypeptide devoid of the alanineresidue.

According to still further features in the described preferredembodiments the polypeptide is an antibody or an antibody fragment.

According to still further features in the described preferredembodiments the antibody or antibody fragment is humanized.

According to still further features in the described preferredembodiments the antibody or the antibody fragment is selected from thegroup consisting of a Fab fragment, an Fv fragment, a single chainantibody and a single domain antibody.

According to still further features in the described preferredembodiments the antibody is produced by a cell hybridoma having thedepository Accession No. CNCM 1-3015 (F8:33 hybridoma) or CNCM 1-3016(F8:33-6-8-10 hybridoma).

According to still further features in the described preferredembodiments the polypeptide is a CDR-containing recombinant polypeptide.

According to still further features in the described preferredembodiments a sequence of the CDR is selected from the group consistingof SEQ ID NOs: 22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, 36, 38, 39,40, 42, 43 and 44.

According to a further aspect of the present invention there is providedan isolated polynucleotide comprising a nucleic acid sequence encodingthe polypeptide.

According to yet a further aspect of the present invention there isprovided an isolated polynucleotide comprising a nucleic acid sequenceencoding the peptide.

According to still a further aspect of the present invention there isprovided a nucleic acid construct comprising the polynucleotide.

According to still further features in the described preferredembodiments the nucleic acid construct further comprising a promoter forregulating expression of the polynucleotide.

According to still a further aspect of the present invention there isprovided an oligonucleotide capable of specifically hybridizing to theisolated polynucleotide and not to a polynucleotide encoding a CD44polypeptide devoid of the alanine residue under stringent hybridizationconditions.

According to still a further aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredientthe polypeptide of and a pharmaceutically effective carrier or diluent.

According to still a further aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredientthe oligonucleotide and a pharmaceutically effective carrier or diluent.

According to still a further aspect of the present invention there isprovided a method of detecting an inflammatory disease in a subjectcomprising detecting in a biological sample of the subject a presenceand/or a level of the polynucleotide, wherein the presence and/or levelof the polynucleotide in the biological sample is indicative of theinflammatory disease in the subject.

According to still a further aspect of the present invention there isprovided a method of detecting an inflammatory disease in a subjectcomprising detecting in a biological sample of the subject a presenceand/or a level of the polypeptide, wherein the presence and/or level ofthe polypeptide in the biological sample is indicative of theinflammatory disease in the subject.

According to still a further aspect of the present invention there isprovided a method of treating an inflammatory disease in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of an agent capable of down-regulatingactivity or expression of the polypeptide, thereby treating theinflammatory disease in the subject.

According to still further features in the described preferredembodiments the agent is selected from the group consisting of:

-   -   (i) an oligonucleotide directed to an endogenous nucleic acid        sequence expressing the polypeptide;    -   (ii) a polypeptide comprising an antigen recognition domain        capable of specifically binding an endogenous amino acid        sequence of the polypeptide; and    -   (iii) a mucosal tolerance-inducing amount of a peptide derived        from the polypeptide.

According to still further features in the described preferredembodiments the inflammatory disease is an autoimmune disease.

According to still further features in the described preferredembodiments the autoimmune disease is rheumatoid arthritis.

According to still a further aspect of the present invention there isprovided a kit for diagnosing an inflammatory disease or apredisposition thereto in a subject, the kit comprising the polypeptideand at least one reagent for detecting complexes including thepolypeptide.

According to still further features in the described preferredembodiments detecting the complexes is effected by an assay selectedfrom the group consisting of immunohistochemistry, ELISA, RIA, Westernblot analysis, FACS analysis, an immunofluorescence assay, and a lightemission immunoassay.

According to still a further aspect of the present invention there isprovided a kit for diagnosing an inflammatory disease or apredisposition thereto in a subject, the kit comprising theoligonucleotide and at least one reagent for detecting hybridization ofthe oligonucleotide.

According to still further features in the described preferredembodiments the at least one reagent is selected suitable for detectinghybridization via an assay selected from the group consisting of PCR,RT-PCR, chip hybridization, RNase protection, in-situ hybridization,primer extension, Southern blot, Northern blot and dot blot analysis.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing CD44 polypeptides,polynucleotides encoding same, antibodies directed thereagainst andmethod of using same for the diagnosis and treatment of inflammatorydiseases.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1A-D show the genomic structure of CD44 (FIG. 1A-B); agarose gelelectrophoresis of primers representing the constant coding region ofCD44 (FIG. 1C); and the partial nucleic acid sequence (exons 4 and 5) ofRA-CD44 and CD44v3-v10, and the corresponding amino acid sequence, whenthe sequences are optionally aligned (FIG. 1D).

FIGS. 2A-B show histograms of fluorescence activated cell sorting (FACS)analysis of different Namalwa cells transfectants (as indicated),incubated with anti-pan-CD44 or anti- CD44v6 (FIG. 2A); the ability ofmAb produced by F8:33 hybridoma to bind, at different concentrations, tothe different Namalwa transfectants was also evaluated (FIG. 2B),wherein the binding of a fluorescein-coupled secondary antibody (IIAb)was used as the control.

FIGS. 3A-C show the results of ELISA (enzyme-linked immunosorbent assay)analysis of F8:33 derived anti-CD44vRA mAb binding to microwells coatedwith soluble CD44vRA, CD44v3-v10, CD44s (CD44vRA-Fc, CD44v3-v10-Fc,CD44s-Fc, respectively), while microwells coated with BSA served as thecontrol (FIG. 3A). Binding of F8:33 derived anti-CD44 mAb was comparedto that of anti-pan-CD44 mAb (FIG. 3B); FIG. 3C shows a Western Blotanalysis of the different CD44 products (CD44s, CD44v3-v10 and CD44vRA,from the left Lanes I, II and III, respectively) immunoblotted with 3G5anti-pan-human-CD44s mAb (Hermes 3, IgG1, ATCC).

FIGS. 4A-B show FACS analysis of the binding of commercial anti-pan-CD44and anti-variant (anti-CD44v6 or anti-CD44v9) mAb to synovial fluidcells from the joint of an RA patient and to primary human keratinocytesprovided from two individuals designated donor M and donor L (FIG. 4A)as well as flow cytometry analysis of the selective binding of F8:33derived anti-CD44vRA (at two concentrations, 4 μg/ml and 2 μg/ml) toprimary keratinocytes from the same two individuals and to synovialfluid cells (FIG. 4B).

FIGS. 5A-B show graphs of cell migration assays performed in thepresence or absence of F8:33 anti-CD44vRA mAb (FIG. 5A) or of anti-panCD44 mAb and according to which Namalwa transfectants were analyzed, inthe absence or the presence of F8:33 anti-CD44vRA mAb (FIG. 5A) oranti-pan CD44 mAb (FIG. 5B) for their ability to cross HA-coated filtersin a transwell migration assay and the percentage of cell migration wascalculated by the number of cells that crossed the membrane in thepresence of antibody, divided by the number of cells that crossed themembrane in its absence, x 100.

FIGS. 6A-B show graphs of cell migration assays performed in thepresence or absence of F8:33 derived anti-CD44vRA mAb (FIG. 6A) or ofanti-pan CD44 mAb (FIG. 6B) using synovial fluid cells from a joint ofan RA patient, or primary keratinocytes, and evaluating their ability tocross HA-coated filters in a transwell migration assay. (The percentageof cell migration was calculated as in FIG. 5B).

FIGS. 7A-E show FACS analysis of the binding of F8:33-6-8-10 derivedanti-CD44vRA mAbs to Namalwa-pcDNA3.1 cells, Namalwa-CD44s cells,Namalwa-CD44v3-v10 cells or Namalwa-CD44vRA cells. The antibody waspresent in the following concentrations: 1.2 mg/ml (FIG. 7A), 120 μg/ml(FIG. 7B), 12 μg/ml (FIG. 7C), 1.2 μg/ml (FIG. 7D) and 120 ng/ml (FIG.7E).

FIG. 8 shows the effect of an anti-CD44vRA mAb (F8:33, squares) onCollagen-induced arthritis (CIA) in mice. Arthritis development wasmonitored for 10 days by measuring paw swelling. The values are themean±SEM. Anti-pan-hCD44 was used as control (triangles).

FIG. 9 shows the effect of KM81 (anti-pan mouse-CD44, squares) and 4D2(control isotype-matched antibody, triangles) on CIA in mice. The valuesare the mean±SEM.

FIGS. 10A-H show FACS analysis of the binding of F8:33 anti-CD44vRAmonoclonal antibody to peripheral blood lymphocytes (PBLs) of healthyindividuals and to synovial fluid cells (SFCs) of an RheumatoidArthritis (RA) patient.

FIGS. 11A-C are agarose gel images showing RT-PCR analyses of CD44variant expression in PBLs from healthy individuals. FIGS. 11A-C areagarose gel images showing RT-PCR analysis of CD44 molecules expressionin PBLs from healthy individuals (11A) and in synovial fluid cells fromRA patients (FIGS. 11B and 11C). FIG. 11B shows RT-PCR analysis usingprimers from constant regions of human CD44 molecule. FIG. 11C showsRT-PCR analysis using sense primer from variant exon 3 (v3).

FIG. 12 is a graph depicting the therapeutic effect of anti CD44vRAantibody on collagen induced arthritis (CIA) of DBA/1 mice. Results aredemonstrated by paw swelling.

FIGS. 13A-J show FACS analysis of the binding of various anti CD44antibodies to arthritic and non-arthritic cells.

FIGS. 14A-D are graphs depicting apoptosis and survival of spleen cellsfrom arthritic and non-arthritic mice treated with anti-CD44vRA mAb(F8:33).

FIG. 15 is a bar graph depicting rheumatoid factor (RF) levels in CIAmice treated with rat anti-mouse cell surface immunoglobulin idiotypeobtained from hybridoma 4D2 (negative control), F8:33 anti-human CD44vRAmAb or KM81-anti-mouse pan CD44 mAb. Levels of RF are indicated prior todisease induction (1) two days following disease onset (2) and 14 daysfollowing disease onset

FIG. 16 is a graph depicting the therapeutic effect of various doses ofanti CD44vRA antibody on arthritic mice as compared to treatment withRemicade™ Results are demonstrated by paw swelling.

FIG. 17 is a bar graph depiciting RF levels in mice treated as describedin FIG. 16.

FIGS. 18A-L are nucleic acid and amino acid sequences of heavy and lightchains of F8:33, F8:33-6-8-10 and MF1-16-11. FIG. 18A depicts thenucleic acid sequence of F8:33 mAb heavy chain (SEQ ID NO: 21). FIG. 18Bdepicts the amino acid sequence of F8:33 mAb heavy chain. CDRs 1, 2, 3(SEQ ID NOs: 22, 23 and 24) as well as Framework regions arehighlighted. FIG. 18C depicts the nucleic acid sequence of F8:33 mAblight chain (SEQ ID NO: 25). FIG. 18D depicts the amino acid sequence ofF8:33 mAb light chain. CDRs 1, 2, 3 (SEQ ID NOs: 26, 27 and 28) as wellas Framework regions are highlighted. FIG. 18E depicts the nucleic acidsequence of F8:33-6-8-10 mAb heavy chain (SEQ ID NO: 29). FIG. 18Fdepicts the amino acid sequence of F8:33-6-8-10 mAb heavy chain. CDRs 1,2, 3 (SEQ ID NOs: 30, 31 and 32) as well as Framework regions arehighlighted. FIG. 18G depicts the nucleic acid sequence of F8:33-6-8-10mAb light chain (SEQ ID NO: 33). FIG. 18H depicts the amino acidsequence of F8:33-6-8-10 mAb light chain. CDRs 1, 2, 3 (SEQ ID NOs: 34,35, 36) as well as Framework regions are highlighted. FIG. 181 depictsthe nucleic acid sequence of MF 1-6-11 mAb heavy chain (SEQ ID NO: 37).FIG. 18J depicts the amino acid sequence of MF 1-16-11 mAb heavy chain.CDRs 1, 2, 3 (SEQ ID NOs: 38, 39 and 40) as well as Framework regionsare highlighted. FIG. 18K depicts the nucleic acid sequence of MF1-16-11 mAb light chain (SEQ ID NO: 41). FIG. 18L depicts the amino acidsequence of MF 1-16-11 mAb light chain. CDRs 1, 2, 3 (SEQ ID NOs: 42,43, 44) as well as Framework regions are highlighted.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of CD44 polypeptides, polynucleotides encodingsame and antibodies and oligonucleotides directed thereagainst which canbe used in the diagnosis and treatment of inflammatory diseases, such asrheumatoid arthritis.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

CD44 is a multistructural and multifunctional cell surface moleculeinvolved in cell proliferation, cell differentiation, cell migration,angiogenesis, presentation of cytokines, chemokines, and growth factorsto the corresponding receptors, and docking of proteases at the cellmembrane, as well as in signaling for cell survival or apoptosis. Thegenomic sequence of human CD44 includes 5 constant exons at the 5′terminus and 5 constant exons at the 3′ terminus, as well as 9 variantexons encompassed therebetween. To date several dozens of splicevariants of CD44 are known.

While reducing the present invention to practice, the present inventorshave uncovered a novel variant mRNA transcript of CD44 (herein CD44-RA,SEQ ID NO: 1) from synovial cells of rheumatoid arthritis (RA) patients.This novel transcript contains the known CD44 constant and variant exonsbut also comprises three additional nucleotides (CAG) that aretranscribed from the end of the intron bridging Exon v4 to Exon v5 andare inserted at the 5′ end of Exon v5. This extra CAG sequence resultsin the insertion of a new codon for the amino acid alanine (SEQ ID NO:2, see FIGS. 1A-D) while leaving the reading frame intact.

While further reducing the present invention to practice, the presentinventors have developed antibodies which specifically bind the CD44variant of the present invention but not other CD44 variants (e.g., SEQID NOs: 3 and 4) and as such can be used in the diagnosis, prognosis,prevention and treatment of RA and other diseases in which the variantCD44 transcript is involved and other disorders which are dependent onthis CD44 variant for onset or progression.

As is illustrated hereinbelow and in the Examples section which follows,the present inventors have successfully shown that a specific antibodyrecognizing CD44vRA is capable of preventing and treatingcollagen-induced arthritis (CIA), in a mouse model of RA. This antibodyexhibited a comparable therapeutic efficacy as compared to a knownanti-RA drug, Remicade™.

Terminology

The isolated polynucleotide described above is also referred to“Rheumatoid Arthritis-CD44 (RA-CD44) variant nucleic acid codingsequence” interchangeably referred to also as the “RA-CD44 variantcoding sequence”, “CD44vRA” or “RA CD44 variant”, refers to nucleic acidmolecules having the sequence shown in SEQ ID NO: 1, nucleic acidmolecules having at least 90% identity (see below) to said sequence andfragments (see below) of the above molecules of , at least 6nucleotides, at least 8 nucleotides, at least 10 nucleotides, at least12 nucleotides, at least 14 nucleotides, at least 16 nucleotides, atleast 18 nucleotides, at least 20 nucleotides long. These moleculescomprise sequences coding for a novel, naturally occurring, alternativesplice variant of the native and known CD44 transcript. It should beemphasized that a novel variant of the present invention is a naturallyoccurring mature mRNA sequence resulting from alternative splicing ofthe primary pre-mRNA transcript and not merely a truncated, mutated orfragmented form of the known sequence.

This RA-CD44 variant sequence comprises Exons 1-5, 15-17 and 19 of theconstant part of the CD44 gene as well as Exons 7-14 (v3-v10) of thevariable region of the gene (Screaton et al, supra). The variant codingsequence comprises three additional bases (CAG) at the 5′ end of Exon v5as explained below.

“RA-CD44 Variant product—also referred at times as “variant product,”“RA-CD44 variant protein,” “variant protein” “RA-CD44 variant peptide”or “CD44vRA variant peptide” or “CD44 variant polypeptide”—is apolypeptide having an amino acid sequence encoded by the RA-CD44 variantcoding sequence. By “polypeptide” is intended a peptide or protein, aswell as peptides or proteins having chemically modified amino acids (seebelow) such as a glycopeptide or glycoprotein. The amino acid sequenceof a preferred RA-CD44 variant product is shown in SEQ ID NO:2 “RA-CD44variant product” also includes homologues (see below) of said amino acidsequence in which one or more amino acids has been added, deleted,substituted (see below) or chemically modified (see below) as well asfragments (see below) of this sequence having at least 6 amino acids.

“Nucleic acid molecule” or “nucleic acid” or “polynucleotide”—asingle-stranded or double-stranded polymer composed of DNA nucleotides,RNA nucleotides or a combination of both types and may include naturalnucleotides, chemically modified nucleotides and synthetic nucleotides.

“An isolated polynucleotide” refers to a single or double strandednucleic acid sequences which is isolated and provided in the form of anRNA sequence, a complementary polynucleotide sequence (cDNA), a genomicpolynucleotide sequence and/or a composite polynucleotide sequences(e.g., a combination of the above).

“Complementary polynucleotide sequence” refers to a sequence, whichresults from reverse transcription of messenger RNA using a reversetranscriptase or any other RNA dependent DNA polymerase. Such a sequencecan be subsequently amplified in vivo or in vitro using a DNA dependentDNA polymerase.

“Genomic polynucleotide sequence” refers to a sequence derived(isolated) from a chromosome and thus it represents a contiguous portionof a chromosome.

“Composite polynucleotide sequence” refers to a sequence, which is atleast partially complementary and at least partially genomic. Acomposite sequence can include some exonal sequences required to encodethe polypeptide of the present invention, as well as some intronicsequences interposing therebetween. The intronic sequences can be of anysource, including of other genes, and typically will include conservedsplicing signal sequences. Such intronic sequences may further includecis acting expression regulatory elements.

“Amino acid sequence”—a sequence composed of any one of the 20 naturallyoccuring amino acids, amino acids which have been chemically modified(see below), or synthetic amino acids.

“Fragment of RA CD44 variant nucleic acid coding sequence”—a fragment ofat least 20 nucleotides of a RA-CD44 variant nucleic acid codingsequence having the sequence of, SEQ ID NO:1, or a fragment of at least20 nucleotides of a RA-CD44 nucleic acid coding sequence having asequence that is 90% identical to the sequence of SEQ ID NO:1, which atleast 20 nucleotides does not appear as a continuous stretch in theoriginal nucleic acid sequence (see below). The fragment will preferablycomprise at least 30 nucleotides, more preferably at least 50nucleotides, most preferably at least 100 nucleotides of the RA-CD44sequence. At a minimum, the fragment will comprise the regioncorresponding to the variant splice junction site which corresponds tonucleotides 908-910 in SEQ ID NO: 1(see explanation below). The fragmentmay be a sequence which was previously described in the context of thepublished CD44 RNA and which affects the amino acid sequence encoded bythe known gene. In the case of the sequence of SEQ ID NO:1, the variantnucleic acid sequence, includes a sequence which was not included in theoriginal CD44, sequence,(a sequence which was in the intron in theoriginal sequence) and the fragment may be that additional sequenceitself.

“Fragment of RA-CD44 variant products”—fragment of at least 6 aminoacids of the RA-CD44 variant polypeptide having the amino acid sequenceof SEQ ID NO:2, or fragments of at least 6 amino acids of a homologue ofsaid polypeptide. The fragments will preferably comprise at least 10amino acids, more preferably at least 20 amino acids, most preferably atleast 30 amino acids, of said RA-CD44 variant (CD44vRA) polypeptide, orsaid homologue at a minimum, the fragment will comprise the regionencoded by the variant splice junction site which corresponds to aminoacid residue 303 in SEQ ID NO:2.

“Homologue of variant”—polypeptide having an amino acid sequence, thatis at least 90% identical to the sequence of SEQ ID NO:2, or at least90% identical to a fragment of at least 6 amino acids of the sequence ofSEQ ID NO:2. The variation in amino acid sequence between the homologueand the sequence of SEQ ID NO:2 or a fragment thereof, arises from theaddition, deletion, substitution or chemical modification of one or moreamino acids of the sequence of SEQ ID NO:2. Where the homologue containsa substitution, the substitution is preferably a conservative one. Theaddition, deletion or substitution may be in regions or adjacent toregions where the RA-CD44 variant product differs from the originalprotein sequence (see below), however, a homologue will preferablyretain the additional alanine residue (corresponding to residue 303 ofSEQ ID NO:2) characteristic of the RA-CD44 variant product.

“Conservative substitution”—refers to the substitution of an amino acidin one class by an amino acid of the same class, where a class isdefined by common physicochemical amino acid side chain properties andhigh substitution frequencies in homologous proteins found in nature, asdetermined, for example, by a standard Dayhoff frequency exchange matrixor BLOSUM matrix. Six general classes of amino acid side chains havebeen categorized and include: Class I (Cys); Class II (Ser, Thr, Pro,Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys);Class V (IIe, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example,substitution of an Asp for another class III residue such as Asn, Gln,or Glu, is a conservative substitution

“Non-conservative substitution”—refers to the substitution of an aminoacid in one class with an amino acid from another class; for example,substitution of an Ala, a class II residue, with a class III residuesuch as Asp, Asn, Glu, or Gln.

“Chemically modified”—when referring to the product of the invention,means a product (protein) where at least one of its amino acid residuesis modified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art. Among the numerous known modificationstypical, but not exclusive examples include: acetylation, acylation,amidation, ADP-ribosylation, glycosylation, glycosaminoglycanation, GPIanchor formation, covalent attachment of a lipid or lipid derivative,methylation, myristlyation, pegylation, prenylation, phosphorylation,ubiqutination, or any similar process. A preferred modification for thepolypeptides of the present invention is pegylation.

“Optimal alignment”—is defined as an alignment giving the highestpercent identity score. Such alignment can be performed using a varietyof commercially available sequence analysis programs, such as the localalignment program LALIGN, using a ktup, of 1 default parameters and thedefault PAM.

“Having at least 90% identity”—with respect to two sets of amino acid ornucleic acid sequences, refers to the percentage of residues that areidentical in the two sequences when the sequences are optimally aligned.Thus, at least 90% amino acid sequence identity means that at least 90%of the amino acids in two or more optimally aligned polypeptidesequences are identical, however this definition explicitly excludessequences which are 100% identical with the original nucleic acidsequence or original protein sequence from which the variant of theinvention was varied.

“Being at least 90% homologous”—refers to an amino acid sequence beingat least 90%, at least 91% homologous, at least 92% homologous, at least93% homologous, at least 94% homologous, at least 95% homologous, atleast 96% homologous, at least 97% homologous, at least 98% homologous,at least 99% homologous, or more, say 100% homologous to SEQ ID NO: 2,as determined using BlastP software of the National Center ofBiotechnology Information (NCBI) using default parameters.

“Expression vector”—refers to vectors that have the ability toincorporate and express heterologous DNA fragments in a foreign cell.Many prokaryotic and eukaryotic expression vectors are known and/orcommercially available. Selection of appropriate expression vectors iswithin the knowledge of those having skill in the art.

The term “oligonucleotide” refers to a single-stranded ordouble-stranded oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally occurring bases, sugars, andcovalent internucleoside linkages (e.g., backbone), as well asoligonucleotides having non-naturally occurring portions, which functionsimilarly to respective naturally occurring portions.

“Deletion”—is a change in either nucleotide or amino acid sequence inwhich one or more nucleotides or amino acid residues, respectively, areabsent as compared to the naturally occurring sequence.

“Insertion” or “addition”—is that change in a nucleotide or amino acidsequence which has resulted in the addition of one or more nucleotidesor amino acid residues, respectively, as compared to the naturallyoccurring sequence.

“Substitution”—replacement of one or more nucleotides or amino acids bydifferent nucleotides or amino acids, respectively as compared to thenaturally occurring sequence. As regards amino acid sequences, thesubstitution may be conservative or non-conservative.

“An isolated polypeptide comprising an antigen recognition domaincapable of speficically binding the CD44 polypeptide of the presentinvention” preferably refers to an antibody or an antibody fragment.

As used herein the term “diagnosing”/“detecting” refers to classifying adisease or a symptom, determining a severity of a disease, monitoringdisease progression, forecasting an outcome of a disease and/orprospects of recovery.

“Biological sample” —The biological sample used in the methods of theinvention can be any appropriate body-derived fluid sample includingwhole blood, peripheral blood monocytes, leukocytes, etc., preferablythe biological sample will comprise synovial fluid cells (synovicytes,SFCs) or synovial fluid, or cellular extracts thereof.

The term “antibody” refers to antibodies of any of the classes IgG, IgM,IgD, IgA, and IgE antibody. The definition includes polyclonalantibodies or monoclonal antibodies. This term refers to wholeantibodies or fragments of the antibodies comprising the antigen-bindingdomain of the anti-variant product antibodies, e.g. scFv, Fab, F(ab′)2,other antibodies without the Fc portion, single chain antibodies,bispecific antibodies, diabodies, other fragments consisting ofessentially only the variable, antigen-binding domain of the antibody,etc., which substantially retain the antigen-binding characteristics ofthe whole antibody from which they were derived. These functionalantibody fragments are defined as follows: (1) Fab, the fragment whichcontains a monovalent antigen-binding fragment of an antibody molecule,can be produced by digestion of whole antibody with the enzyme papain toyield an intact light chain and a portion of one heavy chain; (2) Fab′,the fragment of an antibody molecule that can be obtained by treatingwhole antibody with pepsin, followed by reduction, to yield an intactlight chain and a portion of the heavy chain; two Fab′ fragments areobtained per antibody molecule; (3) (Fab′)2, the fragment of theantibody that can be obtained by treating whole antibody with the enzymepepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′fragments held together by two disulfide bonds; (4) Fv, defined as agenetically engineered fragment containing the variable region of thelight chain and the variable region of the heavy chain expressed as twochains; (5) Single chain antibody (“SCA”), a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule; and (6) Singledomain antibodies are composed of a single VH or VL domains whichexhibit sufficient affinity to the antigen.

The term “substantially retain the antigen binding characteristics ofthe whole antibody” should be understood to mean that the antibodyfragment, derivative or the recombinant antibody molecule specificallybinds the CD44vRA product and that the affinity for the CD44vRA productas determined by Scatchard analysis or biacore analysis (as describedbelow) is at least 30% of the binding affinity of the whole antibody(from which the fragment, derivative or recombinant antibody moleculewas derived). In preferred embodiments, the binding affinity of theantibody fragment, derivative or recombinant antibody molecule for theCD44vRA product is at least 50% of the binding affinity of the wholeantibody.

“Anti-CD44vRA antibody” or “Anti-CD44-RA variant antibody” refers to anantibody that specifically binds to the CD44vRA protein. The antibodymay include polyclonal or monoclonal antibodies, antibody fragments,antibody derivatives and homologues and recombinant antibody moleculesall derived from the monoclonal or polyclonal anti-CD44vRA antibody.Anti-CD44vRA antibodies particularly include the monoclonal antibodies(mAbs) produced by the particular hybridoma described herein (referredto herein by the terms “F8:33 mAb” and “F8:33-6-8-10 mAb” or “MF-1-16-11mAb”) as well as other antibodies that specifically bind to the CD44vRAprotein (referred to as anti-CD44vRA mAbs).

“Recombinant antibody molecule”—refers to an antibody molecule thatresults from manipulation of a monoclonal antibody, typically at thenucleic acid level (i.e., gene, mRNA, etc.), by standard geneticengineering techniques. The recombinant antibody molecule will bereferred to as “derived from” the monoclonal antibody. Recombinantantibody molecules include, for example, chimeric, humanized,primatized, single chain antibodies and fusion proteins. The recombinantantibody molecule substantially retains the antigen bindingcharacteristics of the monoclonal antibody from which it was derived.

“Antibody fragment”—includes a fragment of at least 6 amino acids of theanti-CD44vRA antibody or fragments of at least 6 amino acids of aderivative of said polypeptide. The fragments will preferably compriseat least 10 amino acids, more preferably at least 20 amino acids, mostpreferably at least 30 amino acids, of said anti-CD44vRA antibody, orsaid homologue.

“Derivative of a monoclonal antibody (mAb)”—includes recombinantantibody molecules (as defined above) derived from the original mAb, aswell as mAbs that are chemically modified (as defined above), and alsoincludes mAb labeled with radioactive agents, fluorescent moieties,toxins, antibiotics, etc.

“Homologue of a monoclonal antibody (mAb)”—includes a protein having anamino acid sequence that is at least 90% identical to the sequence ofthe original mAb, or at least 90% identical to a fragment of at least 6amino acids forming the original mAb. The variation in amino acidsequence between the homologue and the original mAb or a fragmentthereof, arises from the addition, deletion, substitution or chemicalmodification of one or more amino acids of the original sequence. Wherethe homologue contains a substitution, the substitution is preferably aconservative one.

“Agonist”—as used herein, refers to a molecule which mimics the effectof the natural RA-CD44 variant product or has enhanced activity comparedwith the natural RA-CD44 variant product, or at times even increases orprolongs the duration of the biological activity of said variantproduct, as compared to that induced by the variant product itself. Themechanism may be by any mechanism known to prolonging activities ofbiological molecules such as binding to receptors; prolonging thelifetime of the molecules; increasing the activity of the molecules onits target; increasing the affinity of molecules to its receptor;inhibiting degradation or proteolysis of the molecules, etc. Agonistsmay be polypeptides, nucleic acids, carbohydrates, lipids, orderivatives thereof, or any other molecules which can positivelymodulate the activity of the variant product.

“Antagonist”—refers to a molecule which inhibits shortens the durationof the biological activity of the natural RA-CD44 variant product. Thismay be done by any mechanism known to deactivate or inhibit biologicalmolecules such as blocking of the receptor, blocking of an active site,competition on a binding site, enhancement of degradation, etc.Antagonists may be polypeptides (e.g., antibodies), nucleic acids,carbohydrates, lipids, or derivatives thereof, or any other moleculeswhich can negatively modulate the activity of said product.

“Treating a disease”—refers to administering a therapeutic substanceeffective to ameliorate symptoms associated with a disease, to lessenthe severity or cure the disease, or to prevent the disease fromoccurring, to prevent the manifestation of symptoms associated with adisease before they occur, to slow down the progression of the diseaseor the deterioration of the symptoms associated therewith, to enhancethe onset of the remission period, to slow down the irreversible damagecaused in the progressive chronic stage of the disease, to delay theonset of said progressive stage, to improve survival rate or more rapidrecovery, or a combination of two or more of the above.

The treatment regimen will depend on the type of disease to be treatedand may be determined by various considerations known to those skilledin the art of medicine, e.g. the physicians. A preferred disease to betreated according to the invention is Rheumatoid Arthritis.

“Detection”—refers, in some aspects of the invention, to a method ofdetection of a disease, disorder, pathological or normal condition. Thisterm may refer to detection of a predisposition to a disease as well asfor establishing the prognosis of the patient by determining theseverity of the disease.

“Probe” also referred to herein as “oligonucleotide (probe)”—a nucleicacid molecule comprising the variant coding sequence, or a sequencecomplementary therewith, when used to detect presence of other similarsequences in a sample. The detection is carried out by identification ofhybridization complexes between the probe and the assayed sequence. Theprobe, in some embodiments, may be attached to a solid support or to adetectable label. The probe will generally be single stranded and willgenerally be between 5 even 10 and 100 nucleotides. The particularproperties of a probe will depend upon the particular use and arereadily within the competence of one of ordinary skill in the art todetermine. Oligonucleotide modifications which are preferably used forin vivo diagnosis and treatment of diseases of the present invention arefurther described hereinbelow.

“Primer pair”—a set of two nucleic acid sequences (“primers”), each ofwhich can serve to prime template-directed polymerization by apolymerase or transcriptase, which primers hybridize to the oppositestrands of a double stranded nucleic acid sequence (“template”) in suchmanner as to direct the polymerization (and amplification) of thedouble-stranded sequence nucleotide sequence located between regions ofprimer hybridization. Such a primer pair can be used in the well knownpolymerase chain reaction (PCR). The design of primers pairs is wellknown in the art and will depend upon the particular sequence to beamplified. In general, the primers are single-stranded, between 10 and40 bases in length and hybridize to regions of the template sequencelocated between 50 and 2000 bases apart.

“Original nucleic acid sequence”—the nucleic acid sequence of the CD44transcript present in synovial cells of non-rheumatoid arthritisindividuals (such as osteoarthritic patients), having exons 1-5, 7-17and 19, with splice junctions as described in Screaton et al. (1992) seeSEQ ID NO: 5, the entire contents of which is incorporated by referenceherein.

“Original protein sequence”—the amino acid sequence of the CD44 proteinencoded by the original nucleic acid sequence (i.e., SEQ ID NO: 6Screaton et al. 1992).

As mentioned and in accordance with the present invention, it has beenfound that synovial cells removed from the joints of rheumatoidarthritis (RA) patients express a variant mRNA transcript of CD44 thatis not found in synovial cells from healthy (that is, non-RA)individuals. This variant CD44 transcript has not previously beendescribed. This CD44 variant will be referred to herein interchangeablyas “RA-CD44 variant nucleic acid coding sequence” or “variant codingsequence” or “RA-CD44” or “RAvCD44”.

In accordance with the invention, it was found that the novel RA-CD44mRNA transcript which contains the constant Exons 1-5, 15-17 and 19 andvariant Exons 7-14 (v3-v10) (as described by Screaton et al., 1992) alsocomprises three additional nucleotides (CAG) that are transcribed fromthe end of the intron bridging Exon v4 to Exon v5 and inserted at the 5′end of Exon v5 in positions 908-910 of SEQ ID NO: 1. This extra CAGsequence results in an insertion of a new codon for the amino acidalanine. The last 3′ original nucleotide “G” of Exon v4 (in position 907of SEQ ID NO:1) together with the new “CA” nucleotides at the 5′end ofthe Exon v5 (positions 908 and 909 of SEQ ID NO:1) form an additionalcodon “GCA” which encodes the amino acid alanine (in position 303 of SEQID NO:2). The translation at both sides of the new insert is not changedas the original “GA T” codon (which encodes the amino acid asparticacid) is preserved (by the new nucleotide “G” in position 910 of SEQ IDNO:1 and the next two original nucleotides “AT” in positions 911 and 912of SEQ ID NO:1) as well as all the other codons of Exon v4 and v5. Thegeneration of the extra CAG in the CD44 transcript from the SFCs of anRA patient is shown FIG. 1A:

As shown in Example 6 of the Examples section which follows, CD44vRA ofthe present invention is detected on synovial fluid cells of RA patients(in about 80% of the 49 patients examined) and to a lower extent (only10%) on the (peripheral blood lymphocytes) PBLs of these patients.CD44vRA is not detected on the PBLs of healthy individuals.Cumulatively, these findings suggest that the expression of CD44vRA isconfined to the inflammation site.

The above findings of a novel RA-CD44 coding variant open the way fordiagnosis, prognosis, prevention and treatment of RA and other diseasesin which the variant CD44 of the invention is involved.

The novel RA-CD44 variant of the invention is a naturally occurringsequence which has not been detected in cells of healthy individuals butonly, in those of individuals suffering from rheumatoid arthritis. TheRA-CD44 variant is presumably produced alternative splicing of theprimary transcript of the known CD44 gene which occurs in cells of suchpatients and does not arise from truncation or mutation of the knownCD44 gene.

The present invention provides by a first aspect, an isolatedpolynucleotide comprising a nucleic acid sequence encoding a CD44variant polypeptide, which comprises, contiguously, a first amino acidsequence being at least 90% homologous to amino acid coordinates 1-302of SEQ ID NO: 2, an alanine residue and a second amino acid sequencebeing at least 90% homologous to amino acid coordinates 304-700 of SEQID NO:2.

The above described isolated polynucleotide being an alternative splicevariant selected from the group consisting of:

(a) a nucleic acid molecule comprising or consisting of the codingsequence SEQ ID NO:1;

(b) a nucleic acid molecule having at least 90% identity to the sequenceof (a) and that differs from the original nucleic acid sequence fromwhich the sequence of (a) was varied;

(c) fragments of (a) or (b) having at least 20 nucleotides andcontaining a sequence which is not present, as a continuous stretch ofnucleotides, in the original nucleic acid sequence from which thesequence of (a) was varied. All of the above nuclei acid molecules willretain the nucleotide region corresponding to nucleotides 908-910 of SEQID NO:1.

The RA-CD44 nucleic acid molecule of the invention additionally includesnucleic acid molecules that are complementary to any of the abovementioned. The isolated polynucleotide/nucleic acid molecule may be inthe form of DNA or in the form of RNA and include DNA, cDNA and genomicDNA and mRNA, synthetic DNA or RNA. The DNA may be doubled stranded orsingle stranded and, if single stranded may be the coding strand or thenon-coding strand.

The RA-CD44 nucleic acid molecule may include the RA-CD44 variant codingsequence only or, alternatively, the coding region may be in combinationwith additional coding sequences such as those coding for fusion proteinor signal peptides. In addition it may be combined with non-codingsequences such as introns and control elements.

Fragments as defined above are also within the scope of the presentinvention. Such fragments, typically comprise at least 20 bases whichcorrespond to a region of the variant coding sequence. Preferably, thefragment comprises at least 30, 40, 50, 60, 70, 80, 90 or 100 or more,nucleotides which correspond to a region of the variant coding sequence.

As mentioned, the novel RA-CD44 transcript discovered by the presentinventors differs from the previously described CD44 transcript by avariation of the splice site junction between exon 8 and exon 9(Screaton et al., supra). This splice site variation results in theinsertion of three additional nucleotides, “CAG” in the sense strand, atthe splice junction between exons 8 and 9. Accordingly, the nucleic acidmolecule of the invention, whether comprising the sequence of SEQ IDNO:1, or a complement or a fragment thereof, or sequences that are, ateleast 90% identical to the sequence of SEQ ID NO:1, or a complement orfragment of thereof, will comprise at a minimum, the three nucleotidescorresponding to the variant splice junction site.

A preferred embodiment, the RA-CD44 nucleic acid molecule comprises thesequence of SEQ ID NO:1 or fragments thereof or sequences having atleast 90% identity to the above sequence as explained above. Inaddition, the RA-CD44 nucleic acid molecule includes nucleic acidsequence coding for the polypeptide of SEQ ID NO:2 or for fragments orhomologues of said polypeptide. The coding sequence may be obtained byany of the methods known in the art. Typically screening of cDNAlibraries using oligonucleotide probes which can hybridize to orPCR-amplify nucleic acid sequences which encode the variant products ofthe invention. A variety of cDNA libraries are commercially availableand the procedures for screening and isolating cDNA clones are withinthe scope of a person skilled in the art. Such techniques are describedin, for example, Sambrook et al., (1989) Molecular Cloning: A LaboratoryManual (2.sup.nd Edition), Cold Spring Harbor Press, Plain View; N.Y.Preferably, the cDNA library is prepared from synovial fluid cells.

The nucleic acid molecules can also be synthetically prepared inaccordance with chemical synthesis methods known in the art.Alternatively, the nucleic acid molecules can be prepared recombinantly.

In addition, the variant nucleic acid molecule of the invention may beprepared by extracting cellular RNA from cells expressing the variantproduct. Such cells may, for example, be synovial fluid cells of RApatients. The RNA is then subjected to reverse transcriptase polymerasechain reaction (RT-PCR) in accordance with methods known in the art suchas those described in Sambrook et al, supra. The amplification productsof the PCR reaction are purified and sequenced.

Due to the degenerate nature of the genetic code, a plurality ofalternative nucleic acid sequences other than that depicted in SEQ IDNO:1 can code for the polypeptide of the invention. Thus, the presentinvention further provides nucleic acid molecules comprising orconsisting of a sequence which encodes the polypeptides of theinvention. Alternative nucleic acid sequences coding for the same aminoacid sequences coded by the sequence of SEQ ID NO:1 are also an aspectof the present invention.

The complete known sequence of the CD44 gene has been published and canbe found for example in Screaton et al, 1992, supra, which alsodescribes the location of exon-intron junctions. Comparison of thecoding sequence of the invention to the known CD44 sequence may becarried out by any of the available computer programs.

The present invention also includes recombinant constructs comprisingone or more of the RA-CD44 variant nucleic acid molecules describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a nucleic acid molecule of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the RA-CD44sequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, and are commercially available. Appropriatecloning and expression vectors for use with prokaryotic and eukaryotichosts are also described in Sambrook, et al., (supra).

The present invention also relates to host cells which comprise vectorsof the invention as well as to the production of the RA-CD44 variantproduct of the invention, by recombinant techniques. Host cells aregenetically engineered (i.e., transduced, transformed or transfected)with the vectors of this invention which may be, for example, a cloningvector or an expression vector. The vector may be, for example, in theform of a plasmid, a viral particle, a phage, etc. The engineered hostcells can be cultured in conventional nutrient media, modified asappropriate for activating promoters, selecting transformants oramplifying the expression of the variant nucleic acid sequence. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to those skilled in the art.

The RA-CD44 variant nucleic acid coding sequences of the presentinvention may be included in any one of a variety of expression vectorsfor expressing a product. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived, from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost. The appropriate RA-CD44 variant DNA sequence may be inserted intothe vector by a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease site(s) byprocedures known in the art. Such procedures and related sub-cloningprocedures are deemed to be within the scope of those skilled in theart.

The RA-CD44 variant coding sequence in the expression vector isoperatively linked to an appropriate transcription control sequence(promoter) to direct mRNA synthesis. Examples of such promoters include:LTR or SV40 promoter, the E. coli lac or tip promoter, the phage lambdaPL promoter, and other promoters known to control expression of genes inprokaryotic or eukaryotic cells or in viruses. The expression vectoralso contains a ribosome binding site for translation initiation, and atranscription terminator. The vector may also include appropriatesequences for amplifying expression. In addition, the expression vectorspreferably contain one or more selectable marker genes to provide aphenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate RA-CD44 variant DNA sequence asdescribed above, as well as an appropriate promoter or control sequence,may be employed to-transfect or transform an appropriate host to permitthe host to express the RA-CD44 variant protein. Examples of appropriateexpression hosts include: bacterial cells, such as E. coli,Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;insect cells such as Drosophila and Spodoptera Sf9; animal cells such asCHO, COS, HEK 293 or Bowes melanoma; adenoviruses; plant cells, etc. Theselection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein. The invention is notlimited by the host cells employed

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., and Battey, I.(1986) Basic Methods in Molecular Biology). Cell-free translationsystems can also be employed to produce polypeptides using RNAs derivedfrom the DNA constructs of the present invention.

The variant products can be recovered and purified from recombinant cellcultures by any of a number of methods well known in the art, includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as necessary, in completing configurationof the mature protein. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps.

The present invention further provides an isolated polypeptidecomprising a CD44 variant polypeptide which comprises, contiguously, afirst amino acid sequence being at least 90% homologous to amino acidcoordinates 1-302 of SEQ ID NO: 2, an alanine residue and a second aminoacid sequence being at least 90% homologous to amino acid coordinates304-700 of SEQ ID NO:2.

According to an embodiment of the present invention the isolatedpolypeptide comprising or consisting of the amino acid sequence of SEQID NO:2 and fragments comprising at least-6 amino acids of the sequenceof SEQ ID NO:2, homologues which are at least 90% identical to thesequence of SEQ ID NO:2, or at least 90% identical to fragments of atleast 6 amino acids thereof, and fragments of the variant product, andits homologues, having at least 10 amino acids. All of the abovepolypeptides, fragments and homologues will retain an alanine residuecorresponding to residue 303 of SEQ ID NO:2.

Due to the fact that the region which differs in the CD44 variant of theinvention as compared to the original sequence is in the 5′ part of exonv5, the homologues of the variant which are within the scope of theinvention should be such which are derivated from the variant by adeletion, addition or substitution of an amino acid either in saidregion or in regions adjacent to it, but excluding deletion orsubstitution of the additional alanine residue.

Thus, the RA-CD44 variant product of the invention is a polypeptideencoded by the RA-CD44 variant coding sequence of the invention. Thepolypeptide has the amino acid sequence of the SEQ ID NO:2, or afragment of at least 6 amino acids thereof, or a sequence homologuehaving at least 90% identity to the sequence of SEQ ID NO:2, or at least90% identity to a fragment of at least 6 amino acids of SEQ ID NO:2,provided that the amino acid sequence is not identical to that of theoriginal protein sequence from which it has been varied. Thepolypeptides, fragments and homologues of the invention will retain thealanine residue corresponding to amino acid 303 of SEQ ID NO:2.

The polypeptide of the invention can be made by any appropriate methodincluding chemical or recombinant synthesis by techniques well known inthe art (described above). Fragments may be obtained by enzymaticdigestion (e.g. using clostrapaine) or chemical (CNBr) digestion of alonger protein. In such a case, the resulting peptides may be separatedby methods known in the art such as by RP-HPLC and the separate peptidesmay then be used for sequencing (e.g. by Euro sequence BV).

The polypeptides in accordance with the invention may also besynthesized by methods known in the art such as on Abbymed 522 by Eurosequence BV. The variant products may also be obtained by recombinantmethods known in the art using the variant coding sequence.

The present invention additionally provides a fusion protein comprisingthe polypeptide having the sequence of SEQ ID NO:2, or fragments orhomologues thereof, fused to another protein or peptide or a chemicalmoiety (e.g., drug, e.g., chemotherapy), as is well known in the art.

In view of the fact that the polypeptide of the invention contains aunique additional region which does not appear in CD44 obtained fromsynovial fluid cells from healthy individuals, antibodies oroligonucleotides [e.g., oligonucleotides which are specifically directedaganist nucleic acid coordinates 890-920 of SEQ ID NO: 1 and Nedvetzkiet al. (2003) J. Clin. Invest. 111:1211-20] specifically directedagainst the RA-CD44 variant product (i.e., protein or RNA, respectively)or peptides derived therefrom may be used specifically for diagnosis,prognosis, prevention and therapy of various diseases in which cellsexpressing this CD44 variant molecule are involved.

By “specifically directed against” or “specifically bind” the RA-CD44variant refers to the ability of an antigen recognition domain (e.g.,such as of an antibody or antibody fragment) to specifically bind theCD44 polypeptide of the present invention (SEQ ID NO: 2) and inabilityto bind a prior art CD44 polypeptide such as selected from the groupconsisting of SEQ ID NO: 4 or 6. For example, the antibody of thepresent invention recognizes and binds to the RA-CD44 variant product inpreference to other known CD44 proteins; in particular the anti-RA-CD44antibodies will recognize and bind to the RA-CD44 variant (CD44vRA)product in preference to the original protein sequence. Generally, theaffinity of anti-CD44vRA antibodies is at least 2-fold greater forbinding to the CD44vRA than for binding to the original protein sequenceor other CD44 isoforms. In a preferred embodiment, the binding affinityof anti-CD44vRA antibodies is at least 10-fold greater for binding tothe CD44vRA than for binding to the original protein sequence or otherCD44 isoforms. The binding affinity may be determined by a Scatchardanalysis on antigens present on cell surfaces, a method that is wellknown in the art (see, for example, Hulmes, E. C. in Receptor-ligandInteractions: a practical approach Chapter 4. Rickwood and Hames, Eds.IRL Press.), as well as by Biacore analysis [e.g., van Regenmortel(2003) Dev. Biol. 112:141-51].

Specific binding may also be inferred from biological assays, such asthe effect of the antibody on cell migration, therapeutic effects, theability to induce cell aggregation, immunofluorescent studies,binding-competition assays, ability to induce apoptosis, effect thereofof CD44vRA signal transduction and the like. In such assays, an antibodywould be defined as CD44vRA specific if the results using this antibodyin the experimental setting differ in a statistically significant mannerfrom those of the control antibody settings.

Thus, by an additional aspect, the present invention provides anisolated polypeptide comprising an antigen recognition domain capable ofspecifically binding a CD44 polypeptide as set forth in SEQ ID NO: 2 andincapable of binding a CD44 polypeptide selected from the groupconsisting of: SEQ ID NO: 4 or 6.

According to a preferred embodiment of this aspect of the presentinvention the isolated polypeptide is an antibody or an antibodyfragment, such as a humanized antibody or antibody fragment.

Examples of antibodies of the present invention are those that areproduced by cell hybridomas having the depository Accession No. CNCM1-3015 (F8:33 hybridoma),CNCM 1-3016 (F8:33-6-8-10 hybridoma) or MF1-16-11 hybridoma.

According to another preferred embodiment of this aspect of the presentinvention, the polypeptide is a complementarity-determining region(CDR)-containing recombinant polypeptide. Such a polypeptide includes atleast one CDR which is sufficient to mediate specific binding of thepolypeptide of this aspect of the present invention to CD44vRA. It willbe appreciated however, that such a polypeptide may include as much asall six CDRs combining the variable domains found in the heavy and lightchains of anti CD44vRA antibodies. Examples of CDR sequences which canbe implemented in the polypeptide are those set forth in SEQ ID NOs: 22,23, 24, 26, 27, 28, 30, 31, 32, 34, 35, 36, 38, 39, 40, 42, 43 and 44.

Preferably, the polypeptides of this aspect of the present invention areneutralizing peptides, which are capable of binding CD44vRA anddown-regulate at least one activity thereof (e.g., cell migration,further described hereinabove).

Anti-RA-CD44 antibodies of the present invention are selected from thegroup consisting of:

(a) antibodies which specifically bind the polypeptide encoded by theRA-CD44 variant nucleic acid coding sequence;

(b) fragments of the antibodies of (a) substantially retaining theantigen binding characteristics of the whole antibody; and

(c) antibodies binding to an antigenic epitope bound by any one of theantibodies of (a) and (b) above.

The antibodies of the invention can be polyclonal antibodies ormonoclonal. By a preferred embodiment, the antibodies of the inventionare such which specifically bind the polypeptide of SEQ ID NO:2.Fragments of such antibodies substantially retaining the antigen-bindingcharacteristics of these antibodies, antibodies binding to an antigenicepitope bound by such antibodies, as well as antibodies which bind to anantigen to which any one of the above Abs specifically bind are alsowithin the scope of the invention. The anti-RA-CD44 antibody of thepresent invention will recognize an epitope of the RA-CD44 variantprotein that is not present in the original protein sequence. Theanti-RA-CD44 antibodies of the invention will preferably recognize anepitope comprising the alanine at residue 303 of SEQ ID NO.2.Alternatively, or in addition, the anti-RA-CD44 antibodies of theinvention will recognize a neoepitope created by a change in the overalltertiary structure of the CD44 protein as a consequence of the alanineresidue insertion at position 303. Such neoepitopes can be, inter alia,conformational epitopes, non-linear epitopes, carbohydrate epitopes orepitopes exposed by differential glycosylation. Epitopes recognized bythe anti-RA-CD44 antibodies of the invention may include epitopes in thevariant region of the peptide sequence at the beginning of the v5 exon,or the epitope may be on a different part of the molecule whose tertiarystructure is altered by the insertion of the alanine residue of thevariant polypeptide. Epitopes may be located in the v5 exon or one ormore other exons, including, for example, the v6 exon. In someembodiments, antibodies that are specific for RA-CD44 do not bind and/orrecognize an antigenic epitope on exon v6. The antibodies of theinvention do not include the antibodies VFF6, VFF4, or VFF7. Antibodiesthat recognize the neoepitope of the RA-CD44 variant protein are usefulin the diagnostic and therapeutic methods described herein. Inaccordance with this embodiment, the antibodies of the invention areused for diagnosis or treatment of infectious and other inflammatorydiseases and autoimmune diseases, most preferably being RA as well asmalignant diseases.

The present invention further provides mouse hybridoma cell lines whichproduce any of the monoclonal Abs of the invention. The hybridomas maybe prepared by any of the methods known in the art (e.g. Kohler, G. andMilstein, C., Nature, 256:495-497,(1975). The present invention furtherprovides recombinant-cell lines or transgenic animals expressing humanor humanized anti-RA-CD44 antibodies of the invention.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (1972)]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single ormore complementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues form acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulingene loci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10,: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

It will be appreciated that targeting of particular compartment withinthe cell can be achieved using intracellular antibodies (also known as“intrabodies”). This technology has been successfully applied in the art(for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).Intrabodies have been shown to virtually eliminate the expression ofotherwise abundant cell surface receptors and to inhibit a proteinfunction within a cell (See, for example, Richardson et al., 1995, Proc.Natl. Acad. Sci. USA 92: 3137-3141; Deshane et al., 1994, Gene Ther. 1:332-337; Marasco et al., 1998 Human Gene Ther 9: 1627-42; Shaheen etal., 1996 J. Virol. 70: 3392-400; Werge, T. M. et al., 1990, FEBSLetters 274:193-198; Carlson, J. R. 1993 Proc. Natl. Acad. Sci. USA90:7427-7428; Biocca, S. et al., 1994, Bio/Technology 12: 396-399; Chen,S-Y. et al., 1994, Human Gene Therapy 5:595-601; Duan, L et al., 1994,Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S-Y. et al., 1994, Proc.Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al., 1994, J. Biol.Chem. 269:23931-23936; Mhashilkar, A. M. et al., 1995, EMBO J.14:1542-1551; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

To prepare an intracellular antibody expression vector, the cDNAencoding the antibody light and heavy chains specific for the targetprotein of interest are isolated, typically from a hybridoma thatsecretes a monoclonal antibody specific for the marker. Hybridomassecreting anti-marker monoclonal antibodies, or recombinant monoclonalantibodies, can be prepared using methods known in the art. Once amonoclonal antibody specific for the marker protein is identified (e.g.,either a hybridoma-derived monoclonal antibody or a recombinant antibodyfrom a combinatorial library), DNAs encoding the light and heavy chainsof the monoclonal antibody are isolated by standard molecular biologytechniques. For hybridoma derived antibodies, light and heavy chaincDNAs can be obtained, for example, by PCR amplification or cDNA libraryscreening. For recombinant antibodies, such as from a phage displaylibrary, cDNA encoding the light and heavy chains can be recovered fromthe display package (e.g., phage) isolated during the library screeningprocess and the nucleotide sequences of antibody light and heavy chaingenes are determined. For example, many such sequences are disclosed inKabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 and in the “Vbase” human germline sequencedatabase. Once obtained, the antibody light and heavy chain sequencesare cloned into a recombinant expression vector using standard methods.

For cytoplasmic expression of the light and heavy chains, the nucleotidesequences encoding the hydrophobic leaders of the light and heavy chainsare removed. An intracellular antibody expression vector can encode anintracellular antibody in one of several different forms. For example,in one embodiment, the vector encodes full-length antibody light andheavy chains such that a full-length antibody is expressedintracellularly. In another embodiment, the vector encodes a full-lengthlight chain but only the VH/CH1 region of the heavy chain such that aFab fragment is expressed intracellularly. In another embodiment, thevector encodes a single chain antibody (scFv) wherein the variableregions of the light and heavy chains are linked by a flexible peptidelinker [e.g., (Gly₄Ser)₃ and expressed as a single chain molecule. Toinhibit marker activity in a cell, the expression vector encoding theintracellular antibody is introduced into the cell by standardtransfection methods, as discussed hereinbefore.

Following is a description of specific hybridomas obtained according tothe teachings of the present invention.

In accordance with the present invention, a hybridoma cell line has nowbeen isolated that produces monoclonal antibodies (mAbs) whichspecifically bind to the

CD44vRA product (CD44vRA) present in synovial fluid cells of individualshaving rheumatoid arthritis (RA) but not present in the synovial fluidcells of non-RA individuals. Because the mAbs are specific for theCD44vRA product in preference to the original, wild type proteinsequence (CD44v3-v10) or other isoforms of CD44, the monoclonalantibodies are useful, inter alia, in a variety of methods requiring theidentification, isolation or targeting of the CD44vRA product.

The present invention further provides mouse hybridoma cell lines havingthe depository Accession Nos. CNCM 1-3015 (for hybridoma F8:33) and CNCM1-3016 (for hybridoma F8:33-6-8-10), (referred to herein as the F8:33and F8:33-6-8-10 hybridoma cell lines), deposited with the CollectionNationale De Cultures De Microorganismes (CNCM), Institut Pasteur,Paris, France, on Apr. 16, 2003) and mAbs produced by said cell line,clones and subclones. The hybridomas may be produced by any of themethods known in the art [e.g. Kohler, G., and Milstein C. Nature256:495-497 (1975)]. The supernatants of the hybridoma cells aretypically screened for antibody binding activity by any one of themethods known in the art such as flow cytometry or by enzyme linkedimmunosorbent assay (ELISA) or radio-immunoassay (RIA). The supernatantscan be screened for production of mAbs capable of binding to any of theCD44vRA products (including fragments, derivatives or homologuesthereof) or to cells expressing said products.

The monoclonal antibodies are typically produced by culturing thehybridoma cells under conditions suitable to produce the monoclonalantibody and isolating the mAb from the cell culture by well knowntechniques. Such conditions and techniques are well known in the art andare described in Mammalian cell biotechnology: a practical approach(Butler, M. Ed, IRL Press). The anti-CD44vRA antibodies of the inventionmay also be produced by recombinant genetic methods well known to aperson skilled in the art; for example, as described in DNA Cloning 4: apractical approach, Chapter 3 (Glover, D. and Hames, B. Eds. IRL Press)and Bebbington et al. [Bio/Technology 10:169 (1992)].

As indicated hereinbefore, the recombinant antibody molecules include,for example, chimeric antibodies [Morrison S. L, Science 229:1202(1985)], humanized antibodies [as described by, for example, Shin S. U.and Morrison S. L., Methods Enzymol., 178:459-476, (1989); Gussow D. andSeemann G., Methods Enzymol. 203:99-121 (1991)], bispecific antibodies[as described by, for example, Weiner L. M. et al., J. Immunol.151:2877-2886 (1993); Goodwin D. A., Int. J. Rad. Appl. Instrum.16:645-651 (1989)], single chain antibodies (scFv, as described by, forexample, gritzapis A. D., et al. Br. J. Cancer 88:1292-1300, (2003)]),complete or fragmentary immunoglobulins [as described by, for example,Coloma M. J., et al., J. Immunol. Methods, 152:89:104, (1992); NesbitM., et al., J. Immunol. Methods, 151:201-208 (1992); Barbas C. F., etal., Proc. Natl. Acad. Sci. USA, 89:10164:10168, (1992)], or antibodiesgenerated by chain shuffling [as described by, for example, Winter G.,et al., Annu. Rev. Immunol., 12:433-455, (1994)]. Humanized antibodiesmay be produced, for example, by CDR grafting (e.g. as described inpublished European patent application No. 0239400). Framework regionsmay also be modified (e.g. as described in European patent applicationNo. 0519596). To humanize antibodies, methods such as PCR (for example,as described in European patent application Nos. 0368684; 0438310; or ininternational patent publication No. WO 92/07075) or computer modeling(for example, as described in international patent publication No. WO92/22653) may be used. Fusion proteins, e.g. single chain antibody/toxinfusion proteins [as described, for example, by Chaudhary V. K., et al.,Proc. Natl. Acad. Sci. USA, 87:9491-9494 (1990); Friedman P. N. et al.,Cancer Res. 53:334-339, (1993)] may also be produced and thus also formpart of the present invention.

The hybridoma cell line of the present invention comprises a nucleicacid encoding the monoclonal antibodies and such nucleic acid is alsowithin the scope of the present invention. Nucleic acid encoding theanti-CD44vRA antibodies can be isolated from hybridoma cell lines bytechniques that are well known in the art including those described inAntibody engineering: a practical approach McCafferty et al., Eds. IRLPress). Such nucleic acids are useful for preparation of additional celllines or transgenic animals expressing the anti-CD44vRA antibodies ormay be used in the preparation of recombinant antibody molecules.

The anti-CD44vRA Abs of the present invention include the monoclonalantibodies expressed by the hybridoma cell line of the presentinvention, whether actually produced in the hybridoma cells or producedby other techniques as are well known in the art; for example, producedin other cell types by transfer of the appropriate genetic material fromthe hybridoma cell (see for example Monoclonal antibodies: the secondgeneration. Zola, H. Ed. BIOS Scientific, Chapters 4-9). Accordingly,there is also provided a monoclonal antibody as defined above, i.e. thatreacts, similarly to the F8:33 derived mAb, with specificity to CD44vRAor to a fragment of said CD44vRA, the CD44vRA fragment comprising anamino acid sequence translated from the region flanking exon v4 to exonv5 of CD44vRA coding sequence or from part of said region and comprisingan Ala residue which is not present in a corresponding fragment ofCD44v3-v10 when CD44vRA and CD44v3-v10 are optimally aligned.

The present invention also concerns homologues, fragments andderivatives (such as chemically modified derivatives, radiolabeledderivatives, derivatives coupled to toxin or antibiotic molecules, andthe like) of the antibodies as defined, all recognizing theantigenically distinct epitope (that includes the additional Ala) andthus substantially retaining the antigen binding specificity of themonoclonal antibodies. Also, recombinant antibody molecules that arederived from the monoclonal antibodies produced by the hybridoma celllines F8:33, F8:33-6-8-10 or MF1-16-11 as well as additional hybridomasproducing anti-CD44vRA mAbs and their clones and subclones thatsubstantially retain the antigen binding characteristics of themonoclonal antibody are explicitly included in the present invention. Itis within the skills of the average artisan to prepare homologues,fragments and derivatives of the antibody of the invention or, startingfrom a sequence analysis of the antibody and/or by use of the hybridomacell line producing this antibody, to prepare recombinant antibodymolecules with the same idiotype, i.e. antibody molecules having thesame amino acid sequence in the region of the antigen binding site(complementarity-determining regions, CDR) as the antibody fromhybridoma cell lines F8:33, F8:33-6-8-10 or MF1-16-11 as well as otherhybridomas producing mAbs recognizing CD44vRA and their clones andsubclones.

As indicated above, the anti-CD44vRA antibody of the present inventionwill recognize an epitope of the CD44vRA product that is not present inthe original, wild type protein sequence (CD44v3-v10). The anti-CD44vRAantibodies of the invention preferably recognize an epitope comprisingthe Ala at residue 303 of SEQ ID NO:2. Alternatively, or in addition,the anti-CD44vRA antibodies of the invention recognize a neo-epitopecreated by a change in the overall tertiary structure of the CD44protein as a consequence of the Ala residue insertion at position 303.Such neo-epitopes can be conformational epitopes, non-linear epitopes,carbohydrate epitopes or epitopes exposed by differential glycosylation.Epitopes may include the variant region of the peptide sequence flankingv5 exon or the epitope may be on a different part of the molecule whosetertiary structure is altered by the insertion of the Ala residue of thevariant polypeptide.

Various hosts can be used for production of antibodies by the hybridomatechnique including rats, mice, etc. The animals may be immunized byinjecting the CD44vRA product (including said fragments, derivatives orhomologues). Various adjuvants may be used to increase the immunologicalresponse such as Freund's, mineral gels, aluminum hydroxide, etc. Theanimals may also be immunized or challenged with cells, for examplehuman Namalwa cells, that have been transfected with vectors carryinggenetic material encoding the CD44vRA products (including fragments,derivatives or homologues thereof) as described herein.

In addition to the hybridoma technique mentioned above, clones andsubclones of this hybridoma as well as continuous cell lines whichproduce antibodies obtained by additional techniques may also be usedsuch as, for example, the EBV-hybridoma technique [Cole et al., Mol.Cell Biol. 62:109 (1984)].

The present invention also provides a nucleic acid molecule comprisingor consisting of a non-coding sequence which is complementary to that ofSEQ ID NO:1 or complementary to a sequence having at least 90% identityto said sequence,(under the conditions defined above) or a fragment ofsaid two sequences (according to the above definition of fragment) alsoreferred to herein as oligonucleotide. The complementary sequence may bea DNA sequence which hybridizes with the sequence of SEQ of ID NO:1 orhybridizes to a portion of that sequence having a length sufficient toinhibit the transcription of the complementary sequence. Thecomplementary sequence may be a DNA sequence which can be transcribedinto an mRNA being an antisense to the mRNA transcribed from SEQ ID NO:1or into an mRNA which is an antisense to a fragment of the mRNAtranscribed from SEQ ID NO:1 which has a length sufficient to hybridizewith the mRNA transcribed from SEQ ID NO:1, so as to inhibit itstranslation. The complementary sequence may also be the mRNA or thefragment of the mRNA itself. Such complementary sequences may be usedfor various diagnostic and therapeutic indications as explained below.

Moderate to stringent hybridization conditions are characterized by ahybridization solution such as containing 10% dextrane sulfate, 1 MNaCl, 1% SDS and 5×10⁶ cpm ³²P labeled probe, at 65° C., with a finalwash solution of 0.2×SSC and 0.1% SDS and final wash at 65° C. andwhereas moderate hybridization is effected using a hybridizationsolution containing 10% dextrane sulfate, 1 M NaCl, 1% SDS and 5×10⁶ cpm³²P labeled probe, at 65° C., with a final wash solution of 1×SSC and0.1% SDS and final wash at 50° C.

Oligonucleotides designed according to the teachings of the presentinvention can be generated according to any oligonucleotide synthesismethod known in the art, such as enzymatic synthesis or solid-phasesynthesis. Equipment and reagents for executing solid-phase synthesisare commercially available from, for example, Applied Biosystems. Anyother means for such synthesis may also be employed; the actualsynthesis of the oligonucleotides is well within the capabilities of oneskilled in the art and can be accomplished via established methodologiesas detailed in, for example: Sambrook, J. and Russell, D. W. (2001),“Molecular Cloning: A Laboratory Manual”; Ausubel, R. M. et al., eds.(1994, 1989), “Current Protocols in Molecular Biology,” Volumes I-III,John Wiley & Sons, Baltimore, Md.; Perbal, B. (1988), “A Practical Guideto Molecular Cloning,” John Wiley & Sons, New York; and Gait, M. J., ed.(1984), “Oligonucleotide Synthesis”; utilizing solid-phase chemistry,e.g. cyanoethyl phosphoramidite followed by deprotection, desalting, andpurification by, for example, an automated trityl-on method or HPLC.

The oligonucleotide of the present invention is of at least 17, at least18, at least 19, at least 20, at least 22, at least 25, at least 30 orat least 40, bases specifically hybridizable with sequence alterationsdescribed hereinabove.

The oligonucleotides of the present invention may comprise heterocylicnucleosides consisting of purines and the pyrimidines bases, bonded in a3′-to-5′ phosphodiester linkage.

Preferably used oligonucleotides are those modified either in backbone,internucleoside linkages, or bases, as is broadly described hereinunder.

Specific examples of preferred oligonucleotides useful according to thisaspect of the present invention include oligonucleotides containingmodified backbones or non-natural internucleoside linkages.Oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example:phosphorothioates; chiral phosphorothioates; phosphorodithioates;phosphotriesters; aminoalkyl phosphotriesters; methyl and other alkylphosphonates, including 3′-alkylene phosphonates and chiralphosphonates; phosphinates; phosphoramidates, including 3′-aminophosphoramidate and aminoalkylphosphoramidates; thionophosphoramidates;thionoalkylphosphonates; thionoalkylphosphotriesters; andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogues ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts, and free acid forms of the above modifications canalso be used.

Alternatively, modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short-chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short-chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene-containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; and 5,677,439.

Other oligonucleotides which may be used according to the presentinvention are those modified in both sugar and the internucleosidelinkage, i.e., the backbone of the nucleotide units is replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example of such an oligonucleotidemimetic includes a peptide nucleic acid (PNA). A PNA oligonucleotiderefers to an oligonucleotide where the sugar-backbone is replaced withan amide-containing backbone, in particular an aminoethylglycinebackbone. The bases are retained and are bound directly or indirectly toaza-nitrogen atoms of the amide portion of the backbone. United Statespatents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262; each ofwhich is herein incorporated by reference. Other backbone modificationswhich may be used in the present invention are disclosed in U.S. Pat.No. 6,303,374.

Oligonucleotides of the present invention may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G) andthe pyrimidine bases thymine (T), cytosine (C), and uracil (U).“Modified” bases include but are not limited to other synthetic andnatural bases, such as: 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and otheralkyl derivatives of adenine and guanine; 2-propyl and other alkylderivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and2-thiocytosine; 5-halouracil and cytosine; 5-propynyl uracil andcytosine; 6-azo uracil, cytosine, and thymine; 5-uracil (pseudouracil);4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, andother 8-substituted adenines and guanines; 5-halo, particularly 5-bromo,5-trifluoromethyl, and other 5-substituted uracils and cytosines;7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine;7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and3-deazaadenine. Additional modified bases include those disclosed in:U.S. Pat. No. 3,687,808; Kroschwitz, J. I., ed. (1990),“The ConciseEncyclopedia Of Polymer Science And Engineering,” pages 858-859, JohnWiley & Sons; Englisch et al. (1991), “Angewandte Chemie,” InternationalEdition, 30, 613; and Sanghvi, Y. S., “Antisense Research andApplications,” Chapter 15, pages 289-302, S. T. Crooke and B. Lebleu,eds., CRC Press, 1993. Such modified bases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines,and N-2, N-6, and O-6-substituted purines, including2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S. et al. (1993),“Antisense Research and Applications,” pages 276-278, CRC Press, BocaRaton), and are presently preferred base substitutions, even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.

Oligonucleotides of the present invention can be used to down-regulateexpression of CD44vRA in a subject in need thereof (further describedhereinbelow), in siRNA protocols, antisense, ribozyme and the like.

The present invention also provides peptides derived from the CD44variant of the present invention. These peptides may be at least about 8amino acids long, at least about 10 amino acids long, at least about 12amino acids long, at least about 14 amino acids long, at least about 16amino acids long, at least about 18 amino acids long, at least about 20amino acids long, at least about 22 amino acids long, at least about 24amino acids long, at least about 26 amino acids long, at least about 28amino acids long, at least about 30 amino acids long (see e.g., SEQ IDNO: 14 and SEQ ID NOs. 15-18). Such peptides may be used for vaccinationor inducing mucosal tolerance which was shown beneficial in thetreatment of auto-immune diseases, such as RA. Such treatments aredescribed, for example, in U.S. Pat. Nos. 5,935,577, 6,019,970,6,790,447, 6,703,361, 6,645,504, 5,961,977, 6,077,509, to Weiner et al.,5,843,449 to Boots et al., and U.S. patent applications Ser. Nos.10/451,370, 10/989,724, 09/944,592, 09/806,400, PCT Nos. IL99/00519 andIL02/00005 and Israel Patent Application No. 126447 to Harats et al.,and in George et al., “Suppression of early atherosclerosis in LDLreceptor deficient mice by oral tolerance with beta2 glycoprotein I”,Cardiovascular Research 2004;62:603-09, (which are incorporated hereinby reference, as if fully set forth). Alternatively, such peptides maybe used for generating antibodies which are specific for CD44vRA.Identification of antibodies which bind specifically to alanine 303 canbe effected using methods known in the art, such as by affinity columnsto which the immunizing peptides are bound and corresponding peptideswhich do not include the alanine are used as control.

Agents of the present invention which are capable of down-regulatingactivity or level of CD44 in a subject in need thereof (e.g., the abovedescribed antibodies, oligonucleotides and peptides) can be used totreat diseases which are dependent on CD44 (activity or expression) fortheir onset or progression, such as for the treatment of inflammatorydiseases, such as, autoimmune diseases, preferably RA.

Thus, the present invention provides a method of treating aninflammatory disease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anagent capable of down-regulating activity or expression of the CD44vRA,thereby treating the inflammatory disease in the subject.

Inflammatory diseases—Include, but are not limited to, chronicinflammatory diseases and acute inflammatory diseases.

Inflammatory diseases associated with hypersensitivity

Examples of hypersensitivity include, but are not limited to, Type Ihypersensitivity, Type II hypersensitivity, Type III hypersensitivity,Type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T lymphocytemediated hypersensitivity and DTH.

Type I or immediate hypersensitivity, such as asthma.

Type II and type III hypersensitivity include, but are not limited to,rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoidarthritis (Krenn V. et al., Histol Histopathol 2000 July ;15 (3):791),spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res2001 ; 3 (3): 189), systemic diseases, systemic autoimmune diseases,systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998 ;17(1-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., ClinDiagn Lab Immunol. 1999 March ;6 (2):156); Chan O T. et al., Immunol Rev1999 June ;169:107), glandular diseases, glandular autoimmune diseases,pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P.Diabetes Res Clin Pract 1996 October ;34 Suppl:S125), thyroid diseases,autoimmune thyroid diseases, Graves' disease (Orgiazzi J. EndocrinolMetab Clin North Am 2000 June ;29 (2):339), thyroiditis, spontaneousautoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec.15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al., NipponRinsho 1999 August ;57 (8):1810), myxedema, idiopathic myxedema (MitsumaT. Nippon Rinsho. 1999 August ;57 (8):1759); autoimmune reproductivediseases, ovarian diseases, ovarian autoimmunity (Garza K M. et al., JReprod Immunol 1998 February ;37 (2):87), autoimmune anti-sperminfertility (Diekman A B. et al., Am J Reprod Immunol. 2000 March ;43(3):134), repeated fetal loss (Tincani A. et al., Lupus 1998 ;7 Suppl2:S107-9), neurodegenerative diseases, neurological diseases,neurological autoimmune diseases, multiple sclerosis (Cross A H. et al.,J Neuroimmunol 2001 Jan. 1 ;112 (1-2):1), Alzheimer's disease (Oron L.et al., J Neural Transm Suppl. 1997 ;49:77), myasthenia gravis (InfanteA J. And Kraig E, Int Rev Immunol 1999 ;18 (1-2):83), motor neuropathies(Kornberg A J. J Clin Neurosci. 2000 May ;7 (3):191), Guillain-Barresyndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J MedSci. 2000 April ;319 (4):234), myasthenic diseases, Lambert-Eatonmyasthenic syndrome (Takamori M. Am J Med Sci. 2000 April ;319 (4):204),paraneoplastic neurological diseases, cerebellar atrophy, paraneoplasticcerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellaratrophies, progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome, polyendocrinopathies, autoimmunepolyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol (Paris)2000 January ;156 (1):23); neuropathies, dysimmune neuropathies(Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl1999 ;50:419); neuromyotonia, acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), cardiovascular diseases, cardiovascular autoimmune diseases,atherosclerosis (Matsuura E. et al., Lupus. 1998 ;7 Suppl 2:S135),myocardial infarction (Vaarala O. Lupus. 1998 ;7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998 ;7 Suppl 2:S107-9),granulomatosis, Wegener's granulomatosis, arteritis, Takayasu'sarteritis and Kawasaki syndrome (Praprotnik S. et al., Wien KlinWochenschr 2000 Aug. 25 ;112 (15-16):660); anti-factor VIII autoimmunedisease (Lacroix-Desmazes S. et al., Semin Thromb Hemost.2000 ;26(2):157); vasculitises, necrotizing small vessel vasculitises,microscopic polyangiitis, Churg and Strauss syndrome,glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis,crescentic glomerulonephritis (Noel L H. Ann Med Interne (Paris). 2000May ;151 (3):178); antiphospholipid syndrome (Flamholz R. et al., J ClinApheresis 1999 ;14 (4):171); heart failure, agonist-likebeta-adrenoceptor antibodies in heart failure (Wallukat G. et al., Am JCardiol. 1999 Jun. 17 ;83 (12A):75H), thrombocytopenic purpura (MocciaF. Ann Ital Med Int. 1999 April-June ;14 (2):114); hemolytic anemia,autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January ;28 (3-4):285), gastrointestinal diseases, autoimmune diseasesof the gastrointestinal tract, intestinal diseases, chronic inflammatoryintestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000January ;23 (1):16), celiac disease (Landau Y E. and Shoenfeld Y.Harefuah 2000 Jan. 16 ;138 (2):122), autoimmune diseases of themusculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist E.et al., Int Arch Allergy Immunol 2000 September ;123 (1):92); smoothmuscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999June ;53 (5-6):234), hepatic diseases, hepatic autoimmune diseases,autoimmune hepatitis (Manns M P. J Hepatol 2000 August ;33 (2):326) andprimary biliary cirrhosis (Strassburg C P. et al., Eur J GastroenterolHepatol. 1999 June ;11 (6):595).

Type IV or T cell mediated hypersensitivity, include, but are notlimited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevittH O. Proc Natl Acad Sci U S A 1994 Jan. 18 ;91 (2):437), systemicdiseases, systemic autoimmune diseases, systemic lupus erythematosus(Datta S K., Lupus 1998 ;7 (9):591), glandular diseases, glandularautoimmune diseases, pancreatic diseases, pancreatic autoimmunediseases, Type 1 diabetes (Castano L. and Eisenbarth G S. Ann. Rev.Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves'disease (Sakata S. et al., Mol Cell Endocrinol 1993 March ;92 (1):77);ovarian diseases (Garza K M. et al., J Reprod Immunol 1998 February ;37(2):87), prostatitis, autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December ;50 (6):893), polyglandular syndrome, autoimmunepolyglandular syndrome, Type I autoimmune polyglandular syndrome (HaraT. et al., Blood. 1991 Mar. 1;77 (5):1127), neurological diseases,autoimmune neurological diseases, multiple sclerosis, neuritis, opticneuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990December ;20 (12):2563), stiff-man syndrome (Hiemstra H S. et al., ProcNatl Acad Sci U S A 2001 Mar. 27 ;98 (7):3988), cardiovascular diseases,cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al., J ClinInvest 1996 Oct. 15 ;98 (8):1709), autoimmune thrombocytopenic purpura(Semple J W. et al., Blood 1996 May 15 ;87 (10):4245), anti-helper Tlymphocyte autoimmunity (Caporossi A P. et al., Viral Immunol 1998 ;11(1):9), hemolytic anemia (Sallah S. et al., Ann Hematol 1997 March ;74(3):139), hepatic diseases, hepatic autoimmune diseases, hepatitis,chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol1990 March ;54 (3):382), biliary cirrhosis, primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November ;91 (5):551), nephricdiseases, nephric autoimmune diseases, nephritis, interstitial nephritis(Kelly C J. J Am Soc Nephrol 1990 August ;1 (2):140), connective tissuediseases, ear diseases, autoimmune connective tissue diseases,autoimmune ear disease (Yoo T J. et al., Cell Immunol 1994 August ;157(1):249), disease of the inner ear (Gloddek B. et al., Ann N Y Acad Sci1997 Dec. 29 ;830:266), skin diseases, cutaneous diseases, dermaldiseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoidand pemphigus foliaceus. Note that several same diseases are can beclassiffied to different classes of hypersensitivity, because theheterogeneity of these diseases.

Examples of delayed type hypersensitivity include, but are not limitedto, contact dermatitis and drug eruption.

Examples of types of T lymphocyte mediating hypersensitivity include,but are not limited to, helper T lymphocytes and cytotoxic Tlymphocytes.

Examples of helper T lymphocyte-mediated hypersensitivity include, butare not limited to, T_(h)1 lymphocyte mediated hypersensitivity andT_(h)2 lymphocyte mediated hypersensitivity.

Autoimmune Diseases

Include, but are not limited to, cardiovascular diseases, rheumatoiddiseases, glandular diseases, gastrointestinal diseases, cutaneousdiseases, hepatic diseases, neurological diseases, muscular diseases,nephric diseases, diseases related to reproduction, connective tissuediseases and systemic diseases.

Examples of autoimmune cardiovascular diseases include, but are notlimited to atherosclerosis (Matsuura E. et al., Lupus. 1998 ;7 Suppl2:S135), myocardial infarction (Vaarala O. Lupus. 1998 ;7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998 ;7 Suppl 2:S107-9), Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S.et al., Wien Klin Wochenschr 2000 Aug. 25 ;112 (15-16):660), anti-factorVIII autoimmune disease (Lacroix-Desmazes S. et al., Semin ThrombHemost.2000 ;26 (2):157), necrotizing small vessel vasculitis,microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focalnecrotizing and crescentic glomerulonephritis (Noel L H. Ann Med InterneParis). 2000 May ;151 (3):178), antiphospholipid syndrome (Flamholz R.et al., J Clin Apheresis 1999 ;14 (4):171), antibody-induced heartfailure (Wallukat G. et al., Am J Cardiol. 1999 Jun. 17 ;83 (12A):75H),thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 April-June;14 (2):114; Semple J W. et al., Blood 1996 May 15 ;87 (10):4245),autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January ;28 (3-4):285; Sallah S. et al., Ann Hematol 1997 March ;74(3):139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al.,J Clin Invest 1996 Oct. 15 ;98 (8):1709) and anti-helper T lymphocyteautoimmunity (Caporossi A P. et al., Viral Immunol 1998 ;11 (1):9).

Examples of autoimmune rheumatoid diseases include, but are not limitedto rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July;15 (3):791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A 1994Jan. 18 ;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al.,Arthritis Res 2001 ; 3 (3): 189).

Examples of autoimmune glandular diseases include, but are not limitedto, pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome diseases include, but are not limited toautoimmune diseases of the pancreas, Type 1 diabetes (Castano L. andEisenbarth G S. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res ClinPract 1996 October ;34 Suppl:S125), autoimmune thyroid diseases, Graves'disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June ;29(2):339; Sakata S. et al., Mol Cell Endocrinol 1993 March ;92 (1):77),spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol2000 Dec. 15 ;165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al.,Nippon Rinsho 1999 August ;57 (8):1810), idiopathic myxedema (Mitsuma T.Nippon Rinsho. 1999 August ;57 (8):1759), ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 February ;37 (2):87), autoimmuneanti-sperm infertility (Diekman A B. et al., Am J Reprod Immunol. 2000March ;43 (3):134), autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December ;50 (6):893) and Type I autoimmune polyglandularsyndrome (Hara T. et al., Blood. 1991 Mar. 1 ;77 (5):1127).

Examples of autoimmune gastrointestinal diseases include, but are notlimited to, chronic inflammatory intestinal diseases (Garcia Herola A.et al., Gastroenterol Hepatol. 2000 January ;23 (1):16), celiac disease(Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16 ;138 (2):122),colitis, ileitis and Crohn's disease.

Examples of autoimmune cutaneous diseases include, but are not limitedto, autoimmune bullous skin diseases, such as, but are not limited to,pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of autoimmune hepatic diseases include, but are not limited to,hepatitis, autoimmune chronic active hepatitis (Franco A. et al., ClinImmunol Immunopathol 1990 March ;54 (3):382), primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November ;91 (5):551; Strassburg C P.et al., Eur J Gastroenterol Hepatol. 1999 June ;11 (6):595) andautoimmune hepatitis (Manns M P. J Hepatol 2000 August ;33 (2):326).

Examples of autoimmune neurological diseases include, but are notlimited to, multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001Jan. 1 ;112 (1-2):1), Alzheimer's disease (Oron L. et al., J NeuralTransm Suppl. 1997 ;49:77), myasthenia gravis (Infante A J. And Kraig E,Int Rev Immunol 1999 ;18 (1-2):83; Oshima M. et al., Eur J Immunol 1990December ;20 (12):2563), neuropathies, motor neuropathies (Kornberg A J.J Clin Neurosci. 2000 May ;7 (3):191); Guillain-Barre syndrome andautoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 April ;319(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. AmJ Med Sci. 2000 April ;319 (4):204); paraneoplastic neurologicaldiseases, cerebellar atrophy, paraneoplastic cerebellar atrophy andstiff-man syndrome (Hiemstra H S. et al., Proc Natl Acad Sci units S A2001 Mar. 27 ;98 (7):3988); non-paraneoplastic stiff man syndrome,progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome and autoimmune polyendocrinopathies (Antoine J C.and Honnorat J. Rev Neurol (Paris) 2000 January ;156 (1):23); dysimmuneneuropathies (Nobile-Orazio E. et al., Electroencephalogr ClinNeurophysiol Suppl 1999 ;50:419); acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al., J NeurolNeurosurg Psychiatry 1994 May ;57 (5):544) and neurodegenerativediseases.

Examples of autoimmune muscular diseases include, but are not limitedto, myositis, autoimmune myositis and primary Sjogren's syndrome (FeistE. et al., Int Arch Allergy Immunol 2000 September ;123 (1):92) andsmooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother1999 June ;53 (5-6):234).

Examples of autoimmune nephric diseases include, but are not limited to,nephritis and autoimmune interstitial nephritis (Kelly C J. J Am SocNephrol 1990 August ;1 (2):140).

Examples of autoimmune diseases related to reproduction include, but arenot limited to, repeated fetal loss (Tincani A. et al., Lupus 1998 ;7Suppl 2:S107-9).

Examples of autoimmune connective tissue diseases include, but are notlimited to, ear diseases, autoimmune ear diseases (Yoo T J. et al., CellImmunol 1994 August ;157 (1):249) and autoimmune diseases of the innerear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29 ;830:266).

Examples of autoimmune systemic diseases include, but are not limitedto, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin DiagnLab Immunol. 1999 March ;6 (2):156); Chan O T. et al., Immunol Rev 1999June ;169:107).

Infectious Diseases

Examples of infectious diseases include, but are not limited to, chronicinfectious diseases, subacute infectious diseases, acute infectiousdiseases, viral diseases, bacterial diseases, protozoan diseases,parasitic diseases, fungal diseases, mycoplasma diseases and priondiseases.

Graft Rejection Diseases

Examples of diseases associated with transplantation of a graft include,but are not limited to, graft rejection, chronic graft rejection,subacute graft rejection, hyperacute graft rejection, acute graftrejection and graft versus host disease.

Allergic Diseases

Examples of allergic diseases include, but are not limited to, asthma,hives, urticaria, pollen allergy, dust mite allergy, venom allergy,cosmetics allergy, latex allergy, chemical allergy, drug allergy, insectbite allergy, animal dander allergy, stinging plant allergy, poison ivyallergy and food allergy.

Cancerous Diseases

Examples of cancer include but are not limited to carcinoma, lymphoma,blastoma, sarcoma, and leukemia. Particular examples of cancerousdiseases but are not limited to: Myeloid leukemia such as Chronicmyelogenous leukemia. Acute myelogenous leukemia with maturation. Acutepromyelocytic leukemia, Acute nonlymphocytic leukemia with increasedbasophils, Acute monocytic leukemia. Acute myelomonocytic leukemia witheosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's;Lymphoctyic leukemia, such as Acute lumphoblastic leukemia. Chroniclymphocytic leukemia; Myeloproliferative diseases, such as Solid tumorsBenign Meningioma, Mixed tumors of salivary gland, Colonic adenomas;Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus,Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovialsarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoidchonodrosarcoma, Ewing's tumor; other include Testicular and ovariandysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignantmelanoma, Mesothelioma, breast, skin, prostate, and ovarian.

Agents (e.g., antibodies, oligonucleotides, peptides) capable ofdown-regualting activity or level of CD44 of the present invention canbe provided to the subject per se, or as part of a pharmaceuticalcomposition where it is mixed with a pharmaceutically acceptablecarrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the agent preparation,which is accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. One of the ingredients included in thepharmaceutically acceptable carrier can be for example polyethyleneglycol (PEG), a biocompatible polymer with a wide range of solubility inboth organic and aqueous media (Mutter et al. (1979).

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, includetransmucosal, transnasal, intestinal or parenteral delivery, includingintramuscular, subcutaneous and intramedullary injections as well asintrathecal, direct intraventricular, intravenous, inrtaperitoneal,intranasal, or intraocular injections.

Alternately, one may administer the preparation in a local rather thansystemic manner, for example, via injection of the preparation directlyinto a specific region of a patient's body. Thus, for example, thepreparation may be directly injected into a joint of an RA patient byintra-articular administration.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro assays. For example, a dose can be formulated in animal modelsand such information can be used to more accurately determine usefuldoses in humans. The estimate of the dosage can be also based bycomparing with other equivalent drugs already in use (e.g., Remicade).

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1].

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions including the preparation of the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

It will be appreciated that the therapeutic agents of the presentinvention can be provided to the individual with additional activeagents to achieve an improved therapeutic effect as compared totreatment with each agent by itself. In such therapy, measures (e.g.,dosing and selection of the complementary agent) are taken to adverseside effects which may be associated with combination therapies.

Administration of such combination therapy can be simulataneous, such asin a single capsule having a fixed ration of these active agents, or inmultiple capsules for each agent.

Thus, for example, the agents of the present invention can beadministered along with nonsteroidal anti-inflammatory drugs (NSAID),disease-modifying antirheumatic drugs (DMARDS), corticosteroids,analgesics, Fibromyalgia medications, chemotherapeutic agents andothers.

In accordance with the diagnostic aspect of the invention, there isprovided a method of detecting an inflammatory disease in a subject. Themethod comprising detecting in a biological sample of the subject apresence and/or a level of the CD44vRA, wherein the presence and/orlevel of the polypeptide in the biological sample is indicative of theinflammatory disease in the subject.

Procedures for obtaining biological samples (i.e., biopsying) fromindividuals are well known in the art. Such procedures include, but arenot limited to, bone biopsy, lymph node biopsy, pleural biopsy, skinbiopsy, thyroid biopsy, CT-guided biopsy, joint biopsy, needleaspiration biopsy and breast biopsy. These and other procedures forobtaining tissue or fluid biopsies are described in details in HypertextTransfer Protocol://World Wide Web (dot) healthatoz(dot)com/healthatoz/Atoz/s earch.asp.

Specifically, a joint biopsy refers to joint or synovial biopsy. In theprocedure a sample of the joint lining or synovial membrane or fluid istaken. Briefly, the procedure is effected in a clinical facility by asurgeon. A number of approaches are available to perform this biopsy:such as through an incision in the joint; with a scope inserted in thejoint; or, more typically, by the insertion of a sharp instrumentthrough the skin. The sample can be taken from any joint, typically theexamined joint is the knee. A sharp instrument (trocar) is pushed intothe joint space. A needle with an attached syringe is inserted into thejoint to withdraw fluid for laboratory analysis. The surgeon may instillanalgesic compounds into the joint and along the needle track before theneedle is withdrawn. The trocar and then the biopsy needle is insertedand specimens taken. After the specimen is taken, both the trocar andthe biopsy needle are removed.

Regardless of the procedure employed, once the biological sample isobtained, the presence of the CD44vRA variant in the sample isdetermined.

As mentioned above, determination of the level of CD44vRA variant in thebiological sample can be effected at the transcriptional level (i.e.,mRNA) using an oligonucleotide probe (such as described above), which iscapable of specifically hybridizing under conditions allowinghybridization to said RA-CD44 variant coding sequence transcript and theformation of detectable probe-transcript hybridization complexes; anddetecting said probe-transcript hybridization complexes, wherein thepresence of said complexes indicates a high probability that the testedindividual from which the sample was obtained has one of the diseases ordisorders involving cells which comprise the RA-CD44 variant codingsequence transcript.

The above example method which is described as a qualitative one (i.e.,presence), may also be quantitative (i.e., level). In accordance withsuch a quantitative method, the level of hybridization complexes inhealthy individuals as well as the level of the complexes formed inindividuals suffering from a specific disorder or disease involvingcells which comprise the RA-CD44 variant RNA transcript is firstdetermined. The level of the complexes detected in the tested individualis then compared to the known calibrated levels of the transcripts.

By quantization of the level of hybridization complexes and calibratingthe quantified results it is possible also to detect the level of thetranscript in the sample.

According to this same aspect, another method is provided foridentifying subject having a disease or disorder involving cells whichcomprise the RA-CD44 variant coding sequence transcript, which methodcomprises the steps of

(a) obtaining a biological sample from the tested individual;

(b) providing a primer pair capable of priming the amplification of aregion of the RA-CD44 variant coding sequence transcript;

(c) contacting the biological sample with the primer pair underconditions allowing amplification of the RA-CD44 variant coding sequencetranscript and the formation of detectable amplification product;

(d) detecting the amplification product, wherein the presence of theamplification product indicates a high probability that the testedindividual from which the sample was obtained has one of the diseases ordisorders involving cells which comprise the RA-CD44 variant codingsequence transcript.

Amplification of the RA-CD44 variant coding sequence transcript canconveniently be accomplished using the well known polymerase chainreaction (PCR) technique (Saiki et al. Science 230: 1350(1995); Mulliset al. Methods Enzymol. 155:335 (1987), Erlich et al. Nature (London)331;461 (1988)). A primer pair is chosen to amplify an appropriateportion of the RA-CD44 variant coding sequence transcript as is wellwithin the competence of one of ordinary skill in the art. The primerpair will typically amplify a portion of the RA-CD44 variant codingsequence transcript in the region in which the variant transcriptdiffers from the original nucleic acid sequence.

By a preferred embodiment, the above diagnostic or prognostic methodsare used for diagnosis or prognosis of RA or for following up thedisease in a tested-indivdual. According to this embodiment, the nucleicacid probe contacted with the sample is such which is able tospecifically detect the presence of an mRNA transcribed from the variantcoding sequence having the sequence of SEQ ID NO:1, or the primer pairis such as to amplify a portion of the mRNA transcribed from the variantcoding sequence having the sequence of SEQ ID NO: 1. In particular, thenucleic acid probe or the primer pair will be such as to allow detectionof the presence of an mRNA transcribed from the variant coding sequencehaving the sequence of SEQ ID NO:1, in preference to detection of anmRNA transcript having the original nucleic acid sequence. In accordancewith the findings of the present invention, a qualitative method may besufficient to identify an individual suffering from RA since the novelcoding sequence has been identified in such patients only. However,various degrees of the disease as well as other related diseases ordisorders may be identified using a quantitative method as describedabove.

It will be appreciated that in accordance with the diagnostic aspect ofthe invention, detection of CD44vRA presence or level can also beeffected at the protein level, such as by using the above-describedantibodies or antibody-fragments. Detection of the antibody-antigencomplexes can be carried out by any of a number of techniques well knownin the art, including, without limitation those described in Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988.

As described with regards to the diagnostic assays based on thedetection of the coding sequence mRNA transcript, in this case as well,the method may be quantitzed to determine the level or amount of thevariant product in the sample which may be indicative of the kind ofdisease or the extent of the disease from which the tested individual issuffering.

By a preferred embodiment, the above diagnostic method is used foridentifying an individual having a disorder or disease involving cellswhich express the RA-CD44 variant product. However, the method may alsobe used for identifying an individual having a disease involving cellswhich express isoforms of the CD44 protein other than the RA-CD44variantof the invention.

As indicated above, this method as well can be quantitized to determinethe level or the amount of the RA-CD44 variant product in the sample,alone or in comparison to the level of the original protein sequence.

The diagnostic reagents described hereinabove can also be included inkits. Such kits comprise an additional aspect of the invention.Diagnostic kits, for example, would typically include one or more of theAbs of the invention, a conjugate of a specific binding partner for theAbs, a label capable of producing a detectable signal and directions forits use. A kit for diagnosing predisposition to, or presence of adisease associated with the CD44vRA of the present invention in asubject can include an antibody (e.g., labeled) of the present inventionin a one container and a solid phase for attaching multiple biologicalsamples packaged in a second container with appropriate buffers andpreservatives and used for diagnosis.

The following summarizes methods of determining levels of biomolecules(i.e., RNA or protein) of interest in biological samples.

The expression level of the RNA in the cells of the present inventioncan be determined using methods known in the art.

Northern Blot Analysis

This method involves the detection of a particular RNA in a mixture ofRNAs. An RNA sample is denatured by treatment with an agent (e.g.,formaldehyde) that prevents hydrogen bonding between base pairs,ensuring that all the RNA molecules have an unfolded, linearconformation. The individual RNA molecules are then separated accordingto size by gel electrophoresis and transferred to a nitrocellulose or anylon-based membrane to which the denatured RNAs adhere. The membrane isthen exposed to labeled DNA probes. Probes may be labeled usingradioisotopes or enzyme-linked nucleotides. Detection may be performedby autoradiography, colorimetric reaction, or chemiluminescence. Thismethod allows for both quantitation of an amount of a particular RNAmolecule and determination of its identity by a relative position on themembrane which is indicative of a migration distance in the gel duringelectrophoresis.

RT-PCR Analysis

This method uses PCR amplification of relatively rare RNA molecules.First, RNA molecules are purified from cells and converted intocomplementary DNA (cDNA) using a reverse transcriptase enzyme (such asan MMLV-RT) and primers such as oligo-dT, random hexamers, orgene-specific primers. Then by applying gene-specific primers and TaqDNA polymerase, a PCR amplification reaction is carried out in a PCRmachine. Those of ordinary skill in the art are capable of selecting thelength and sequence of the gene-specific primers and the PCR conditions(i.e., annealing temperatures, number of cycles, and the like) that aresuitable for detecting specific RNA molecules. It will be appreciatedthat a semi-quantitative RT-PCR reaction can be employed, by adjustingthe number of PCR cycles and comparing the amplification product toknown controls.

RNA In Situ Hybridization Stain

In this method DNA or RNA probes are attached to the RNA moleculespresent in the cells. Generally, the cells are first fixed tomicroscopic slides to preserve the cellular structure and to prevent theRNA molecules from being degraded, and then are subjected tohybridization buffer containing the labeled probe. The hybridizationbuffer includes reagents such as formamide and salts (e.g., sodiumchloride and sodium citrate) which enable specific hybridization of theDNA or RNA probes with their target mRNA molecules in situ whileavoiding non-specific binding of probe. Those skilled in the art arecapable of adjusting hybridization conditions (i.e., temperature,concentration of salts and formamide, and the like) for specific probesand types of cells. Following hybridization, any unbound probe is washedoff and the slide is subjected to either a photographic emulsion, whichreveals signals generated using radio-labeled probes, or to acolorimetric reaction, which reveals signals generated usingenzyme-linked labeled probes.

In Situ RT-PCR Stain

This method is described by: Nuovo, G. J. et al. (1993). Intracellularlocalization of polymerase chain reaction (PCR)-amplified hepatitis CcDNA. Am J Surg Pathol 17, 683-690); and Komminoth, P. et al. (1994)Evaluation of methods for hepatitis C virus detection in archival liverbiopsies. Comparison of histology, immunohistochemistry, in situhybridization, reverse transcriptase polymerase chain reaction (RT-PCR)and in situ RT-PCR. Pathol Res Pract 190, 1017-1025). Briefly, theRT-PCR reaction on fixed cells involves the incorporation of labelednucleotides in the reaction. The reaction is effected using a specificin situ RT-PCR apparatus, such as the laser-capture microdissectionPixCell II™ Laser Capture Microdissection (LCM) system available fromArcturus Engineering (Mountainview, Calif., USA).

Oligonucleotide Microarray

In this method, oligonucleotide probes capable of specificallyhybridizing with the polynucleotides of the present invention areattached to a solid surface (e.g., a glass wafer). Each oligonucleotideprobe is of approximately 20-25 nucleic acids in length. To detect theexpression pattern of the polynucleotides of the present invention in aspecific cell sample (e.g., blood cells), RNA is extracted from the cellsample using methods known in the art (using, e.g., a TRIZOL® solution,Gibco-BRL™, USA). Hybridization can take place using either labeledoligonucleotide probes (e.g., 5′-biotinylated probes) or labeledfragments of complementary DNA (cDNA) or RNA (cRNA). Briefly,double-stranded cDNA is prepared from the RNA using reversetranscriptase (RT) (e.g., Superscript™ II RT), DNA ligase, and DNApolymerase I, all according to the manufacturer's instructions(Invitrogen Life Technologies, Frederick, Md., USA). To prepare labeledcRNA, the double-stranded cDNA is subjected to an in vitro transcriptionreaction in the presence of biotinylated nucleotides using, e.g., theBioArray™ HighYield™ RNA Transcript Labeling Kit (Enzo Diagnostics,Inc., Farmingdale, N.Y., USA). For efficient hybridization the labeledcRNA can be fragmented by incubating the RNA in 40 mM Tris Acetate (pH8.1), 100 mM potassium acetate, and 30 mM magnesium acetate, for 35minutes at 94° C. Following hybridization, the microarray is washed andthe hybridization signal is scanned using a confocal laser fluorescencescanner, which measures fluorescence intensity emitted by the labeledcRNA bound to the probe arrays.

For example, in the Affymetrix® GeneChip® Microarray (Affymetrix, Inc.,Santa Clara, Calif., USA), each gene on the array is represented by aseries of different oligonucleotide probes, of which each probe pairconsists of a perfect-match oligonucleotide and a mismatcholigonucleotide. While the perfect-match probe has a sequence exactlycomplimentary to the particular gene, thus enabling the measurement ofthe level of expression of the particular gene, the mismatch probediffers from the perfect match probe by a single base substitution atthe center base position. The hybridization signal is scanned using theAgilent DNA Microarray Scanner™ (Agilent Technologies, USA) and theMicroarray Suite™ (MAS) (Affymetrix, Inc.) software subtracts thenon-specific signal of the mismatch probe from the signal resulting fromthe perfect-match probe.

Alternatively, expression level of proteins expressed in cells can bedetermined using methods known in the art. Activity of CD44vRA can bedetermined using a cell migration assay such as described in theExamples section which follows.

Enzyme-Linked Immunosorbent Assay (ELISA)

This method involves fixation of a sample containing a protein substrate(e.g., fixed cells or a proteinaceous solution) to a surface such as awell of a microtiter plate. A substrate-specific antibody coupled to anenzyme is applied and allowed to bind to the substrate. Presence of theantibody is then detected and quantitated by a colorimetric reactionemploying the enzyme coupled to the antibody. Enzymes commonly employedin this method include horseradish peroxidase and alkaline phosphatase.If well calibrated and within the linear range of response, the amountof substrate present in the sample is proportional to the amount ofcolor produced. A substrate standard is generally employed to improvequantitative accuracy.

Western Blot

This method involves separation of a substrate from other protein bymeans of an acrylamide gel followed by transfer of the substrate to amembrane (e.g., nitrocellulose, nylon, or PVDF). Presence of thesubstrate is then detected by antibodies specific to the substrate,which are in turn detected by antibody-binding reagents.Antibody-binding reagents may be, for example, protein A or secondaryantibodies. Antibody-binding reagents may be radiolabeled orenzyme-linked, as described hereinabove. Detection may be byautoradiography, colorimetric reaction, or chemiluminescence. Thismethod allows both quantitation of an amount of substrate anddetermination of its identity by a relative position on the membraneindicative of the protein's migration distance in the acrylamide gelduring electrophoresis, resulting from the size and othercharacteristics of the protein.

Radioimmunoassay (RIA)

In one version, this method involves precipitation of the desiredprotein (i.e., the substrate) with a specific antibody and radiolabeledantibody-binding protein (e.g., protein A labeled with I¹²⁵) immobilizedon a precipitable carrier such as agarose beads. The radio-signaldetected in the precipitated pellet is proportional to the amount ofsubstrate bound.

In an alternate version of RIA, a labeled substrate and an unlabelledantibody-binding protein are employed. A sample containing an unknownamount of substrate is added in varying amounts. The number of radiocounts from the labeled substrate-bound precipitated pellet isproportional to the amount of substrate in the added sample.

Fluorescence-Activated Cell Sorting (FACS)

This method involves detection of a substrate in situ in cells bound bysubstrate-specific, fluorescently labeled antibodies. Thesubstrate-specific antibodies are linked to fluorophores. Detection isby means of a cell-sorting machine, which reads the wavelength of lightemitted from each cell as it passes through a light beam. This methodmay employ two or more antibodies simultaneously.

Immunohistochemical Analysis

This method involves detection of a substrate in situ in fixed cells bysubstrate-specific antibodies. The substrate specific antibodies may beenzyme-linked or linked to fluorophores. Detection is by microscopy, andis either subjective or by automatic evaluation. With enzyme-linkedantibodies, a colorimetric reaction may be required. It will beappreciated that immunohistochemistry is often followed bycounterstaining of the cell nuclei, using, for example, Hematoxyline orGiemsa stain.

The present invention also envisages a method for identifying candidateagonist or antagonist compounds of the RA-CD44 variant productcomprising: providing a polypeptide comprising an amino acid sequencesubstantially as depicted in SEQ ID NO:2, or a fragment of such asequence; contacting a tested candidate compound with said polypeptide;measuring the effect of said candidate compound on the activity of saidpolypeptide and selecting those compounds which show at least 70%,preferably 90%, reduction of the level or duration of said activity(antagonists) or at least 70%, preferably 90%, increase in the level orduration of said activity (agonists). Any assay measuring a knownactivity of CD44 may be used to test the effect of the candidatecompound such as for example, cell adhesion, rolling, extravasation ormigration of cells.

Wherein the candidate agonist is one that has an activity which isessentially identical to the activity of RA-CD44, the method foridentifying it will comprise the following steps: providing apolypeptide comprising an amino acid sequence substantially as depictedin SEQ ID NO:2, or a fragment of such a sequence; measuring the activityof said polypeptide in a test assay; measuring the activity of saidcandidate compound in said test assay; comparing the above measuredactivities, wherein an activity measured for the candidate being atleast 70%, preferably 90%, of the activity measured for the polypeptudeindicating that said candidate compound is a compound having an activitywhich is essentially identical to the activity of the RA-CD44 variantproduct.

The test assay in the above methods may be any assay in which theactivity of RA-CD44 may be measured such as for example, the assaysmeasuring activities mentioned above.

The present invention also concerns compounds identified by the abovemethods.

In accordance with yet another aspect of the invention, peptide ligandswhich bind to the RA-CD44 variant product of the invention areidentified. The identification of such ligands may be carried out, forexample, by using phage display peptide libraries. Methods involving useof such libraries are described, for example in Nissim A.; EMBO J.,13:692 (1994) The phage libraries used may be antibody libraries as wellas peptide libraries.

Thus a method is provided for the identification of a peptide whichbinds to the RA-CD44 variant product comprising: incubating cellsexpressing the RA-CD44 variant product with a phage display peptidelibrary; washing the cells to remove unbound phages; eluting bound phagefrom the cells; amplifying the resulting bound phage; determining thedisplay peptide sequence of the bound phage.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Cloning and Expression of CD44-RA Variant Generation ofAntibodies There Against and Effect Thereof on Cell Migration andArthritis Progression Materials and Experimental Procedures

Cloning and transfection of human CD44vRA, CD44v3-10 and CD44s —Forcloning human CD44vRA cDNA, the total synovial fluid cell population ofRA patients undergoing joint aspiration was isolated. RNA was separatedwith a commercial kit (Promega, Madison, Wis.), CD44vRA cDNA wasprepared by RT-PCR (PTC-100™ Programmable Thermal Controller, MJResearch, Watertown, Mass.), using the following primers representingthe constant coding regions of CD44 (FIG. 1A):

Ex1-sense, (SEQ ID NO: 7) 5′-GAATTCGCCG CCACCATGGA CAAGTTTTGG TGG-3′;Ex19-antisense, (SEQ ID NO: 8) 5′-TCTAGATTAC ACCCCAATCT TCATG-3;

PCR product size was confirmed by agarose gel electrophoresis andsequencing (ABI PRISM 310, Perkin-Elmer, Wellesley, Mass.) and PstI (NewEngland BioLabs, Beverly, Mass.) digestion (the nucleotide insertion inCD44vRA introduces a PstI digestion site).

The PCR product was excised from the gel, purified and subcloned into apGEM vector (Promega). Positive clones were selected by white/bluescreening. Plasmids were purified with a commercial kit (Promega),subjected in EcoRI/Xbal-double digestion and cloned into the pcDNA3.1vector (Invitrogen, Paisley, UK) in which the gene product wasexpressed. The plasmid was transfected into the CD44-negative NamalwaBurkett lymphoma cell line (ATCC, Manassas, Va., ATCC No: CRL-1432) asdescribed [Zhang Z et al. J. Biol. Chem. 276:41921-42929 (2001)]. Forcloning of human CD44v3-10, RNA was isolated from human keratinocytes(Hadassah University Hospital, Jerusalem), and for cloning of humanCD44s, RNA was isolated from the HeLa cervical cancer cell line(obtained from ATCC, Manassas, Va., ATCC No: CCL-2), using theabove-described protocol. Transfection of CD44v3-v10 and CD44s cDNAs aswell as of the pcDNA3.1 vector (“empty vector”) was performed asdescribed above. Accordingly, the transfected Namalwa cells weredesignated Namalwa-CD44vRA, Namalwa-CD44v3-v10, Namalwa-CD44s andNamalwa-Neo (empty), respectively.

Preparation of soluble hCD44v3-10, hCD44vRA and hCD44s plasmids—Thesoluble CD44v3-10 cDNA was cloned from total RNA of primary humankeratinocyte by RT-PCR amplification, using two primers assigned fromthe published CD44 sequence;

Ex1s: (SEQ ID NO: 9) 5′-TATCTAGAGC CGCCACCATG GACAAGTTTT GGTGG-3′Ex16/17as: SEQ ID NO: 10) 5′-TATCTAGAGCC ATTCTGGAAT TTGGGGTGT-3′

Both primers contained a Xbal recognition site. The PCR products weredigested with Xbal enzyme and pCXFc zeovector was digested with NheIenzyme. After digestion, the PCR products were ligated into the pCXFczeovector to generate CD44v3-v10-immunoglobulin (Ig)-Fc recombinant.Using the same protocol, the soluble CD44vRA and soluble CD44s cDNAswere cloned from synovial cells of rheumatoid arthritis patients. Thesoluble CD44 fragments were assigned from the published sequence of CD44(1-1824 bases) [Screaton, G. R., et al., (1992) ibid.].

Transient transfection of the soluble CD44 plasmids into 293T cells—Aquantity of 3 μg of each one of the above-indicated Fc containingplasmids was incubated for 20 min with 12 μl of FuGene (Roche). Themixture was added into 15 cm cell plates containing 70% confluent 293Tcells. Supernatant was collected after 48 h and 72 h. The CD44-Ig Fcfragmented proteoglycans were purified on protein-G column and analyzedfor their accurate size by SDS-PAGE and immunoblotting withanti-pan-CD44 mAb (Hermes-3, ATCC Manassas, Vs., ATCC No: HB-9480).

Reverse transcriptase polymerase chain reaction (RT-PCR)—RNA wasextracted from synovial fluid cells of RA patients, primary humankeratinocytes or Namalwa-CD44vRA cells, using RNA-BEE reagent (RNAisolation solvent, Tel-Test Inc., Friendswood, Tex.) according to themanufacturer's instructions. Reverse transcription was performed with 5units of AMV reverse transcriptase (Promega, Medison, Wis.) in a 20_(i)ll reaction volume containing 50 mM Tris-HCl, pH 8.3, 50 mM KCL, 10mM MgCl₂, 10 mM dithiothreitol (DTT) and 20 units RNasin (Promega,Medison, Wis.), using 500 ng of RNA and 100 ng of oligo d(T)18 primer(Promega, Medison, Wis.). Reaction samples were incubated for 1 hour at41° C. and then the reverse transcriptase was inactivated by heating themixture for 10 minutes at 65° C. The amplification was performed in amicro-processor-controlled incubator (MiniCycler™, MJ Research,Watertown, Mass.), using 0.5 μl of the reverse transcriptase reactionproduct (cDNA) in a final volume of 50 μl containing 50 mM KCl, 1.5 mMMgCl₂, 10 mM Tris-HCl, pH 9.0, 250 μM dNTPs and 2.5 units Taq DNApolymerase (Promega, Medison, Wis.). The following primers (FIG. 1A)were added to reaction mixture:

hs5′ sense: 5′-GATGGAGAAAGCTCTGAGCATC-3′; (SEQ ID NO: 11) pv3I sense:5′-ACGTCTTCAAATACCATCTC-3′; (SEQ ID NO: 12) hs3′ anti-sense:5′-TTTGCTCCACCTTCTTGACTCC-3′; (SEQ ID NO: 13)

The CD44 amplification was carried out for 30 cycles with denaturationat 94° C. for 1 minute, annealing at 50° C. for 1 minute and extensionat 72° C. for 2 minutes, followed by 10 minutes final extension at 72°C. The amplified products were resolved on 1.5% agarose gel.Determination of the cellular CD44 isoform transcripts was based on theposition of the band in relation to the markers' ladder, and on theexpected by size of the different CD44 variants.

Generation of monoclonal antibody secreting Hybridomas—Thirty merCD44vRA peptide: SNPEVLLQTT TRMTADVDRNGTTAYEGNWN (SEQ ID NO:14) obtainedfrom Corixa (Seattle, Wash.), and/or 100 μg/ml soluble CD44vRA producedas described above, both emulsified in complete Freund's adjuvant (CFA)(Sigma), were used to immunize subcutaneously or intramuscularly8-week-old female C75BL/6 mice. The immunization was repeated on days 14and 28 and two weeks later the mice were bled and their sera were testedby flow cytometry for their ability to bind to Namalwa cells expressingCD44. The animals with highest polyclonal anti-CD44 antibody titers wereselected and boosted intraperitoneally (i.p.) with 10⁸ Namalwa-CD44vRAcells. After 72 h, spleen cells from the mice were harvested and fusedwith SP 2/0 myeloma cells according to Kohler and Milstein [Kohler, G.,and Milstein C. Nature 256:495-497 (1975)]. After one day of incubationin enriched RPMI 1640 (Sigma) containing L-glutamine,penicillin-streptomycin solution, sodium pyruvate and MEM-eaglenon-essential amino acids (Biological Industries Ltd., Israel) and 20%fetal bovine serum (FBS) (Sigma), the hybridomas were grown inClonaCell™-HY Hybridoma Selection Medium (medium D, StemCellTechnologies Inc.). Between days 10 to 14, isolated hybridoma colonieswere collected from the semi-solid agar and grown in 96-well plates(Costar) in enriched RPMI 1640 medium containing HAT media supplement(Sigma) and 20% of FBS. At day 7 after plating, the supernatants fromisolated hybridoma clones were screened by flow cytometry for theirability to bind to Namalwa-Neo, Namalwa-CD44v3-10 or Namalwa-CD44vRAcells. Hybridoma whose supernatants bound selectively or preferentiallyto Namalwa-CD44vRA were cloned by limiting dilution and then re-clonedfor additional three cycles. The isolated hybridoma were maintained inenriched RPMI 1640 containing HAT media supplement and 20% FBS. Theisotype of the CD44vRA-positive hybridoma supernatant was determined byELISA using Clonotype System-HRP (Southern Biotechnology Associates,Inc.).

Fluorescence activated cell sorting (FACS) analysis—A quantity of 10⁶cells were incubated with 3G5 anti-pan-CD44s (Hermes 3, IgG1) F-10-44-2anti-pan-CD44 mAb (IgG2b, Serotec, Oxford, UK, known also as anti-CD44smAb) or VFF7 anti-CD44v6 mAb (IgG1, Bender MedSystem, Vienna, Austria)for 45 min on ice. After extensive washing, the cells were incubatedwith fluorescein isothiocyanate (FITC)-conjugated secondary anti-Igantibody (Jackson ImmunoResearch, West Grove, Pa.) for 30 min on ice.The cells were then washed and analyzed with a Flow Cytometer (BecktonDickinson, San Jose, Calif.).

RF analysis—Was effected using ELISA commercial kit: mouse rheumatoidfactor cat. no. 6200 of Wirostats Inc., USA according to Manufacturerinstructions.

Enzyme-linked immunosorbent assay (ELISA)—Polystyrene plates of 96 wells(Nunc) were coated with purified CD44vRA-IgFc, CD44v3-v10-IgFc,CD44s-IgFc soluble proteins or with BSA (100 μg/ml per well diluted in100 μl sodium acetate buffer, pH 7.0). After overnight incubation at 4°C., the plates were washed three times with phosphate-buffered saline(PBS), pH 7.4, containing 0.05% Tween 20 (PBS/T). Following blockingwith 10% milk in PBS at 37° C. for 2 h, different concentrations ofF8:33 anti-CD44vRA mAb or anti-pan-CD44 (Hermes 3) mAb were added to thewells. The plates were incubated at 37° C. for 1 h, washed and asecondary goat anti-mouse polyvalent peroxidase-conjugated antibody(Jackson ImmunoResearch) was added for an additional 1 h. The enzymereaction was developed with 0.04% H₂O₂ and 0.04% O-phenylenediamine inphosphate-citrate buffer, pH 5.0. The optical density was measured at405 nm on a microplate reader MRX (Dynatech Laboratories) and valuesabove 0.100 were considered positive.

Western Blot analysis—Cells were lysed in NP-40 buffer and 100 μg ofproteins were run on denaturing SDS-PAGE and transferred to a PVDFmembrane (Millipore, Bedford, Mass.). Blots were blocked with 1% BSA inPBS containing 0.1% Tween-20 (PBS-T), and incubated for 1 h with 1 μg/mlHermes-3 anti-pan-CD44 mAb, which was obtained from the ATCC hybridoma(ATCC No: HB-9480) supernatant and purified on a protein-G column. Theblots were rewashed in PBS-T, incubated with the appropriateHRP-conjugated anti-Ig secondary antibody (1:10,000 dilution) (JacksonImmunoResearch) for 45 min, rewashed in PBS-T and developed with ECLreagent (Amersham Biosciences, Buckinghamshire, UK).

Transwell migration assay—Migration assays were performed in transwellplates (Costar, Cambridge, Mass.) of 6.5 mm diameter. The upper andlower compartments of the transwells were separated by a 5 μM porepolycarbonate filter coated overnight at 4° C. with 0.5 mg/ml hyaluronicacid (HA; H1876, Sigma) in PBS, and then washed 3 times with RPMI 1640.A quantity of 5×10⁵ transfected Namalwa cells or human primarykeratinocytes suspended in RPMI 1640 was added to the upper compartmentand 293 T cell supernatant diluted in RPMI or stromal cell-derivedfactor-1 (SDF-1) (400 ng/ml diluted in RPMI; R&D systems) was added tothe lower compartment. To evaluate the anti-migratory capacity of theantibody, F8:33 anti-CD44vRA mAb or anti-pan-CD44 (F10-44-2) mAb wasadded, in different concentrations, to the transwell plates, which werethen incubated at 37° C. with 5% CO₂ for 4 hours. Cells that migrated tothe lower compartment were counted at the end of the incubation periodby fluorescence-activated cell sorter (FACS; Becton Dickinson, San JoseCalif.) on high speed for one minute and values of cell number/min wererecorded.

Inhibition of Collagen-induced arthritis in mice—The following mAbs wereused: rat anti-mouse CD44 constant region (rat, IgG2b) obtained fromhybridoma KM81 (ATCC, TIB-241;22); rat anti-mouse cell surfaceimmunoglobulin idiotype (rat, IgG2b) obtained from hybridoma 4D2(provided by J. Haimovich, Tel-Aviv University, Maloney et. al.,Hybridoma 4:191-209, 1985) was used as an irrelevant isotype control forKM81 mAb; mouse anti-human CD44vRA (mouse, IgG2a) obtained fromhybridoma F8:33 (our developed hybridoma); mouse anti-human CD44constant region (mouse, IgG2a) obtained from hybridoma F10-44-2 (waspurchase from Serotec Company) was used as a non-biofunctional isotypecontrol for F8:33 mAb.

In order to induce arthritis in mice, Male DBA/1 mice (8-12 weeks old)were injected intradermally at the base of the tail with 200 μg type IIcollagen purified from bovine articular cartilage and emulsified incomplete Freund's adjuvant (CFA; Difco Laboratories, Detroit, Mich.,USA) as described in Williams et. al., Proc. Natl. Acad. Sci. USA89:9784-9788, 1992. The mice received a booster injection of 200 μg typeII collagen emulsified in CFA, 3 weeks after the first dose. The micewere inspected daily and each animal with erythema and/or swelling inone or more limbs was randomly assigned to one of 4 groups, whichreceived intraperitoneal (i.p.) injections of KM81 anti-mouse CD44 mAb,4D2 isotype matched control mAb, F8:33 anti-human CD44vRA mAb orF10-44-2 non-biofunctional isotype control anti-human CD44 mAb. Eachmouse was injected on the day of disease onset (day 0) and then everyother day for 10 days with 200 μg antibody in 100 μl PBS. Arthritis wasmonitored over the 10 days treatment period by measuring paw swelling.In order to measure paw swelling, the thickness of each affected hindpaw was measured with microcalipers. The results are expressed as adirect measure of paw width in millimeters.

Statistical analysis—Data were analyzed using microcomputer programs forone-way ANOVA, followed by Student's t-test for unpaired values. P<0.05was considered significant. The results are expressed as the mean±s.e.m. Each experiment was repeated at least 3 times, all showingsimilar results.

Example 1 mRNA of CD44 Expressed on Synovial Fluid Cells of RheumatoidArthritis (RA) Patients Contains an Intron-Derived Extra Trinucleotide

Synovial fluid cells from RA patients were isolated following jointaspiration. Their total RNA was reverse transcribed and subjected toPCR, using primers representing the constant coding regions of CD44.

FIG. 1B shows the RT-PCR of synovial fluid cells derived from the jointof representative RA patients. Two major signals were detected: afast-migrating band (571 bp) corresponds to CD44s, and a slow-migratingband (1714 bp) corresponds to CD44v3-v10, which is also expressed onkeratinocytes. These findings were confirmed by direct sequencing (datanot shown).

A CD44 variant was detected in synovial fluid cells derived from 52 of55 RA patients and from 12 of 14 patients with psoriatic arthritis(PSA). Five of 12 samples from synovial fluid cells of osteoarthritis(OA) patients also displayed the CD44 variant.

The CD44 variant RT-PCR products from 29 of 55 RA patients and 8 of 14PSO patients were sequenced. An extra trinucleotide (CAG) was detectedbetween exon v4 and exon v5 (FIG. 1C) in 23 (of 29) RA patients and 7(of 8) PSA patients following computerized alignment versus the wildtype variant-CD44v3-v10 (FIG. 1C). The CAG trinucleotide was transcribedfrom the extreme end of the intron bridging exon v4 to exon v5,precisely at the splicing junction. This trinucleotide allows thetranslation of alanine (Ala) without interfering with the entire readingframe of CD44 transcript. Rheumatoid arthritis-derived CD44 variant withextra CAG was designated CD44vRA.

Example 2 Production of Anti-CD44vRA mAb

Expression of extra Ala in CD44vRA appeared to induce a configurationchange, allowing the generation of mAbs able to discriminate between theRA variant and the wild-type isoform-CD44v3-v10 (or the standard isoformCD44s). CD44vRA, CD44v3-v10 (derived from human keratinocytes) and CD44s(derived from HeLa cells) cDNAs were transfected into CD44-negativeNamalwa Burkett lymphoma cell line. CD44 transfectants expressing highlevels of CD44s, as well as CD44v3-v10 and CD44vRA transfectantsexpressing equal levels of v6-containing CD44 variant were selected(FIG. 2A). The transfectants were designated Namalwa-CD44s,Namalwa-CD44v3-v10 and Namalwa-CD44vRA, respectively. Namalwa cellstransfected with an empty vector were designated Namalwa-Neo.

C57BL/6 mice were immunized with soluble CD44vRA, incorporated into

CFA and challenged with Namalwa-CD44vRA cells as described in Materialsand Methods. Splenocytes from mice showing polyclonal anti-CD4vRAantibodies in their serum were fused with SP2/0 myeloma cells. Hybridomacell clones were selected according to the ability of their supernatantsto bind to Namalwa-CD44vRA, but substantially not to Namalwa-CD44v3-v10,Namalwa-CD44s or to Namalwa-Neo, as indicated by flow cytometry. Clonesand sub-clones were established from positive hybridoma cell colonies,and they were stable in culture for over 8 months. Anti-CD44vRA mAbsfrom supernatants of positive hybridomas, designated F8:33, werepurified on G protein column. Flow cytometry further revealed (FIG. 2B)that at a concentration of 0.4 μg/ml, F8:33 anti-CD44vRA mAb interactedwith Namalwa-CD44vRA, but not with the other transfectants, includingthe wild type-CD44v3-v10. At a concentration of 2 μg/ml or higher(Table 1) F8:33 cross-reacted with Namalwa-CD44v3-v10 (FIG. 2B) and ateven higher concentrations (>100 μg/ml)—with Namalwa-CD44s andNamalwa-Neo cells as well (not shown).

TABLE 1 Binding of F8:33 anti-CD44vRA mAb to Cells F8:33 mAb Namalwa-Namalwa- RA synovial Human Concentration CD44vRA CD44v3-10 fluid cellsKeratinocytes 0.2 μg/ml + − + − 0.4 μg/ml + − + −  2 μg/ml + + + −  4μg/ml + + + −  20 μg/ml + + + −  40 μg/ml + + + − 100 μg/ml  + + + − 200μg/ml  + + + + + binding; − no binding; N.D. not done

The above findings were confirmed by ELISA showing that F8:33 mAb bound,in a dose-dependent manner, to CD44vRA-coated microwells at higher ratesthan to CD44v3-v10 or CD44s-coated microwells (FIG. 3A). In contrast,anti-pan CD44 mAb bound to a similar extent to CD44vRA and CD44v3-v10(FIG. 3B), while it did not bind to CD44s, presumably due to itsinability to recognize the relevant epitope. The identity of the solubleCD44 proteins was verified by Western Blot (FIG. 3C).

The antibodies bound to CD44vRA coated on microwells or expressed onNamalwa cells at higher rates than to the corresponding wild typemolecule-CD44v3-v10 or to CD44s. Notably, CD44vRA and CD44v3-v10 areexpressed to a similar extent on Namalwa cells, while CD44s is expressedon these cells at an even higher level, indicating that the preferentialbinding of F8:33 to Namalwa-CD44vRA is not quantitatively dictated. Theselective binding of F8:33 anti-CD44vRA mAb to Namalwa-CD44vRA wasdetected at concentrations equal to or lower than 0.4 μg/ml. Atincreasing concentrations, F8:33 first cross-reacts withNamalwa-CD44v3-v10 and then with Namalwa-CD44s, implying differentialbinding affinity, the highest to cell surface CD44vRA and the lowest tocell surface CD44s.

Example 3 Selective Targeting of RA Synovial Fluid Cells by F8:33Anti-CD44vRA mAb

The Namalwa transfectants are, in fact, an artificial model forevaluating the binding capacity and bioactivity of anti-CD44vRA mAbs. Toobtain a more realistic assessment, the interaction of F8:33 withprimary RA synoviocytes and primary keratinocytes was examined.Keratinocytes were chosen as a reference group, because they are knownexpressors of CD44v3-v10, the wild type counterpart of CD44vRA. Flowcytometry analysis revealed slightly higher expression of CD44s onsynovial fluid cells of an RA patient than on keratinocytes derived fromtwo donors, with variations in expression of v6-containing CD44. It wasalso noted that keratinocytes expressed v9-containing CD44 molecules (inwhich CD44v3-v10 is included) at much higher levels than RA synoviocytes(FIG. 4A). Even so, at concentrations of 2 and 4 μg/ml, F8:33anti-CD44vRA mAb interacted with synovial fluid cells, but not withkeratinocytes as indicated by flow cytometry (FIG. 4B). Even at as higha concentration as 100 μg/ml, F8:33 selectively bound to RAsynoviocytes, while at a concentration of 200 μg/ml F8:33 cross-reactedwith keratinocytes (Table 1). Synovial fluid cells were identified asCD44vRA-positive cells by Pstl digestion of their cDNA. Keratinocytesconstitutively express CD44v3-v10.

Example 4 The CD44vRA-Dependence of Transwell Cell Migration

The question whether the interaction between F8:33 anti-CD44vRA mAb andRA synovial fluid cells displays a bioactivity was investigated. To thisend, the ability of F8:33 anti-CD44vRA to inhibit the migration ofNamalwa transfectants as well as RA synovial fluid cells andkeratinocytes in transwell migration assay was determined.

F8:33 anti-CD44vRA reduced, in a dose-dependent manner, the ability ofNamalwa-CD44vRA cells to cross the HA-coated membrane more effectivelythan that of Namalwa-CD44v3-v10 (FIG. 5A). This antibody could notdisplay any inhibitory effect on the migration of Namalwa-CD44s andNamalwa-Neo cells (at a concentration of 1 μg/ml, F8:33 enhanced themigration of Namalwa-Neo cells). Isotype-matched anti-pan-CD44 mAb(recognizing a constant epitope on all CD44 isoforms) slightlyinfluenced, at the highest concentration only, the migration of all CD44transfectants (FIG. 5B), implying the selective anti-migratory effect ofF8:33.

F8:33 anti-CD44vRA mAb also reduced, in a dose-dependent manner, theability of CD44vRA-positive RA synovial fluid cells, but notkeratinocytes, to cross the HA-coated membrane as indicated by transwellmigration assay (FIG. 6A). In contrast, the anti-pan-CD44 mAb did notinterfere with the cell migration of both cell types (FIG. 6B).

F8:33-6-10-8 is also highly specific for the CD44vRA product (FIGS.7A-7E). Antibodies derived from the F8:33-6-10-8 hybridoma wereincubated with Namalwa cells expressing empty vector (Namalwa-pcDNA3.1),standard CD44 (Namalwa-CD44std.), CD44v3-v10 product(Namalwa-CD44v3-v10) or CD44vRA product (Namalwa-CD44vRA). Already at aconcentration of 1.2 μg/ml (FIG. 7D), the F8:33-6-10-8 antibodies reactspecifically with the CD44vRA expressing cells, whereas they do not bindto the other cells. Even at a concentration of 1.2 mg/ml (FIG. 7A), theF8:33-6-10-8 antibodies bind to the CD44vRA-expressing cells with ahigher affinity than to the other cells.

Example 5 Inhibition of Collagen-Induced Arthritis in Mice

The anti-CD44vRA, F8:33 mAb was tested in the mouse in vivo assay ofcollagen-induced arthritis (CIA). Collagen-induced arthritis (CIA) isthe animal analogue of rheumatoid arthritis (RA), a recurrent, systemicdisease characterized by chronic inflammation within the joints,associated with synovitis and erosion of cartilage and bone.

As can be seen in FIG. 8, Collagen-induced arthritis (CIA) is inhibitedby treatment with the anti-CD44vRA mAb F8:33, as indicated by reductionin paw swelling. In contrast, the control antibody, anti-pan human CD44monoclonal antibody F10-44-2, was not able to reduce the arthriticactivity.

As a positive control, KM81, anti-pan mouse CD44 antibody was used (FIG.9). This antibody was also able to reduce paw swelling caused by CIA. Incontrast, 4D2 monoclonal antibody, a mouse isotype-matched unrelatedmAb, which was used as a negative control, did not change the course ofCIA.

To summarize, F8:33 shows of in vivo biological activity, as indicatedby its ability to reduce arthritic activity in mice.

Example 6 Expression Analysis of CDvRA

Expression of CDvRA in PBLs was analyzed as follows.

Materials and Experimental Procedures

Flow cytometry analysis of F8:33 anti-CD44vRA mAb binding to PBL ofhealthy individuals—Peripheral white blood cells of 62 healthy donorswere isolated by a Ficoll gradient and analyzed by flow cytometry, usingcommercial anti-human-pan-CD44 mAbs (Hermes 3 and F10-44-2) andanti-human CD44v6 mAb, as well as F8:33 anti-CD44vRA, as describedabove.

RT-PCR—Effected as described above.

Results

PBLs from healthy donors express only the standard CD44 (CD44s), asindicated by their immunostaining with Hermes 3 and F10-44-2 (FIGS.10A-B). Anti-human v6 mAb did not bind to the normal PBL (FIG. 10C),indicating that these cells do not express v6-containing CD44 variants.Furthermore, F8:33 did not bind to normal PBL, even at the highestconcentration (100 μg/ml; FIG. 10D) used. Synovial fluid cells from RApatients served as positive control for immunostaining with F8:33. FIGS.10E-H clearly shows similar expression of CD44 on both normal PBLs andRA synovial fluid cells, as indicated by staining withanti-human-pan-CD44 mAbs. Further, RA synovial fluid cells (but notnormal PBLs) express CD44v6, as indicated by staining with anti-humanCD44v6 mAb (FIG. 10G). F8:33 anti-CD44vRA mAb bound exclusively to thesynovial fluid cells of RA patients (FIG. 10H).

The above described FACS results were further substantiated by RT-PCRanalysis. As shown in FIGS. 11A-C, only CD44s transcripts were detectedin normal PBLs (representative results from 12 healthy individuals areshown in FIG. 11A). In contrast, the mRNA of RA synovial fluid cells (6representative samples) contained the expected CD44s, as shown whenprimers from the constant regions were used (FIG. 11B) and a CD44variant of CD44vRA size, as shown when primers from variant regions wereused (FIG. 11C).

In conclusion, CD44vRA was detected on synovial fluid cells of RApatients (in about 80% of the 49 patients examined) and to a lowerextent (only 10%) on the PBLs of these patients. CD44vRA was notdetected on the PBLs of healthy individuals. Cumulatively, thesefindings suggest that the expression of CD44vRA is confined to theinflammation site.

Example 7 Therapeutic Effect of F8:33 Anti-Human CDvRA

Experimental Procedures

As stated above, CIA is considered an animal model of human RA. Thejoint inflammatory disease was induced by two subcuntaneous (s.c.)injections (into the base of the tail) of type II collagen at three weekintervals. Joint inflammation, which appears several days after the lastcollagen injection, causes enhanced foot pad swelling, as well asswelling of other joints. The thickness of nonarthritic footpads is 1.5to 1.7 mm. A footpad thickness of 1.9 to 2.0 mm was arbitrarily set asdisease onset. Footpad thickness gradually increased, reaching a plateauat day 8. Antibodies (200 μg per injection) were injected at diseaseonset and then every other day for 12 days. The antibody effect onfootpad thickness was determined by a double blind assay, using anelectronic microcaliper to measure the thickness of the joints. The micewere bled prior to disease induction, following disease onset and upontermination of the experiment (around day 12). The sera were stored andlater analyzed for the presence of rheumatoid factor (RF). The mice weresacrificed upon termination of the experiment and joints were removedfrom some of the animals for histopathological examination,immunohistochemical staining and RT-PCR analysis.

Results

The results of the clinical experiment, which included 3 groups, 10 micein each group, were recorded and shown in FIG. 12. As shown, injectionof KM81 anti-mouse-pan-CD44 mAb (positive control), which recognizes theconstant CD44 epitope, markedly reduced arthritic inflammatory jointswelling in the DBA/1 mice, as previously shown by the present inventors(J. Autoimmun. 13, 39-47, 1999). Injection of F8:33 anti-human CD44vRAmAb also reduced the arthritic activity in the DBA/1 mice, albeitslightly less than KM81 (the difference was, however, insignificant).Injection of isotype-matched control antibodies (negative control: 4D2served as control for KM81, F10-44-2 served as control for F8:33) didnot influence the course of the disease in the DBA/1 mice.

In conclusion, anti-CD44vRA, F8:33 antibody, reduced joint inflammationby almost 80% when compared with the negative control group. Its effectwas close to that of the KM81 anti-mouse mAb (positive control), showingits efficient bioactivity.

The above-described clinical results were further substantiated byhistopathological examinations. The rear footpad joints of 2 arthriticmice treated with isotype-matched control mAb (negative control), 2arthritic mice treated with KM-81 anti-mouse-pan CD44 mAb (positivecontrol) and 2 arthritic mice treated with F8:33 anti-human-CD44vRA mAb,were histopathologically examined (Patho-Lab, Kiryat Weizmann, Rehovot).The pathological report (not shown) confirmed the clinical findingsdescribed here in above and in FIG. 13. The mouse joints from arthriticmice treated with KM81 and F8:33 recovered, at least partly, whencompared with joints from arthritic mice treated with isotype-matchedcontrol mAbs (negative control).

Flow cytometry analysis of F8:33 binding to spleen cells from arthriticand nonarthritic mice. As is evident from FIG. 5, F8:33 bound to spleencells from arthritic mice, but not to spleen cells from nonarthriticmice, whereas anti-human-pan CD44 mAb (Hermes 3) and anti-mouse pan-CD44mAb (KM81) bound to both cell types. Anti-mouse-CD44v6 mAb showedmarginal binding to arthritic cells, but did not bind to nonarthriticcells (FIG. 5). In the context of these findings it should be stressedthat arthritic splenocytes express both CD44s and v6-containing CD44variants, as indicated by FACS analysis and RT-FCR, whereas nonarthriticsplenocytes express standard CD44 only. The above results suggest thatF8:33 exclusively identifies and binds to spleen cells of arthriticmice.

The ability of F8:33 to induce apoptosis of spleen cells was thendetermined. For this purpose, a two-dimensional flow cytometryimmunostaining with annexin V and propidium iodide was effected. Thefindings shown in FIGS. 14A-D exhibit a correlation between the abilityof increasing concentrations of F8:33 to bind to arthritic splenocytes(FIGS. 11 and 12) and their ability to induce apoptosis (FIGS. 14A-D).Furthermore, F8:33 induced apoptosis (FIG. 14A) or decreased survival(FIG. 14B) of arthritic splenocytes (from mice with CIA), but not ofnonarthritic splenocytes (FIGS. 14C and D). In contrast, anti-pan-CD44mAbs (directed against either human or mouse CD44) did induce apoptosisor even protected the splenocytes from apoptosis (see FIG. 14C). Inconclusion, F8:33 anti-CD44vRA mAb shows in vitro bioactivity, asimplied by its ability to induce apoptosis selectively in arthriticsplenocytes. In contrast, anti-pan-CD44 mAbs does not exhibit suchselectivity.

The expression of Rheumatoid Factor (RF) was then analyzed as follows.Randomly selected mice from the different treatment groups were bledprior to disease induction with collagen (FIG. 15, bars marked by 1),two days following onset of disease (or the first day of antibodytreatment) (FIG. 15, bars marked by 2), as well as 14 days followingdisease onset, i.e., upon termination of the experiment (FIG. 15, barsmarked by 3). The sera were analyzed, using a commercial kit, for RFlevels, an accepted assay for human RA diagnosis. The findings confirmedthe clinical data relating to footpad swelling, as shown in FIG. 12 andthe histopathological analysis. Thus, injection of F8:33 anti-humanCD44vRA mAb or KM81-anti-mouse-pan CD44 mAb reverted or preserved thepreimmunization levels of RF detected in the sera before treatment withthe antibody. After injection of isotype-matched control mAbs the levelsof RF were enhanced (FIG. 15).

The above-described clinical results were validated at various kineticsand doses of F8:33 and the therapeutic efficacy of the antibody wascompared to that of a known anti-RA drug, anti-human TNF mAb (Ramicade).

43 mice included in this experiment were divided into 5 experimentalgroups and the results were recorded (FIG. 16). Systemic injection(starting at CIA onset) of anti-mouse TNFα mAb (5 mice) or anti-humanTNFα mAb (3 mice), adhering to the standard protocol (i.e., 200 μgantibody/mouse every other day for 12 days) markedly reduced the CIAactivity in DBA/1 mice (total, 8 mice), as indicated by tracing footpadswelling. Systemic injection of F8:33 anti-CD44vRA mAb according to thestandard protocol (i.e., first injection at disease onset) substantiallyreduced the CIA activity in the DBA/1 mice (9 mice, F8:33 200 μg),confirming the above-described clinical findings (FIG. 12). Systemicinjection of 70 μg per dose of F8:33, instead of 200 μg per dose, didnot influence the course of CIA in the DBA/1 mice (9 mice, F8:33 70 μg).Systemic injection of F8:33, starting at full blown CIA, rather than atdisease onset, reduced joint inflammation in the DBA/1 mice (7 mice;F8:33 200 μg per dose), albeit at a later phase of the disease.Furthermore, on day 12 identical reduction of inflammation was recordedfor both groups of mice. Systemic injection of 4D2 isotype-matchedcontrol mAb for KM81 (5 mice) and F10-44-2 isotype-matched controlanti-pan-CD44 mAb for F8:33 (5 mice) did not influence the course of CIAin the DBA/1 mice.

In conclusion, F8:33 anti-CD44vRA was as effective as anti-TNFα mAb.Furthermore, this antibody reduced CIA activity even when injectedalmost at the peak of the joint inflammatory response. Finally, itshould be noted that the therapeutic dose of F8:33 (200 μg perinjection) is equivalent to the therapeutic dose of Remicade in RApatients (10 mg/kg).

The RF level in sera of of mice tested as shown in FIG. 16 wasdetermined. As shown in FIG. 17, F8:33, anti-mouse TNFα and Remicade(bars marked by 3) caused the RF to revert to the pre-immunizationlevels detected in the sera of the individual mice before treatment withthe antibody (bars marked by 1), whereas isotype-matched control mAb(anti-human-pan CD44 mAb) did not affect the enhanced levels of RFdetected shortly after disease onset (bars marked by 2), thussubstantiating the clinical data.

The therapeutic effect of F8:33 was challenged in a different mousemodel, which enables, unlike the conventional CIA model, thesynchronized appearance of arthritis and the unlimited use of all mousestrains. In this experiment BALB/c mice were subjected to a cocktail offour different anti-collagen monoclonal antibodies and two days laterthey were inoculated i.p. with LPS. The mice simultaneously developedarthritis, as indicated by footpad swelling. Injection of F8:33 afterdisease onset (5 mice) significantly reduced the arthritic inflammationwhen compared with that in mice that received PBS (4 mice, data notshown).

Example 8 Cloning of the Heavy (V_(H)) and Light (V_(L)) Chains ofF8:33, F8:33-6-8-10 and MF 1-16-11 Murine mAbs

The CDRs of F8:33, F8:33-6-8-10 and MF1-16-11 murine mAbs were clonedand sequenced.

Experimental Procedures

Approximately 1-2 million mAb-secreting hybridoma cells were collectedand total RNA was isolated using RNAsol kit according to themanufacturer's instructions (BioLab). cDNA was produced by reversetranscription, using RNA template, an 18 base oligo-dT primer andSuperScript II reverse transcriptase (Invitrogen). Reaction samples wereincubated at 42° C. for 1 hour. The resulting cDNA was used as atemplate for PCR amplification using Taq polymerase (Invitrogen) and mixof various 5′ and 3′ primers (Pharmacia Biotec) specific for murineV_(H) and V_(L) genes. PCR reaction samples were incubated at 94° C. for2 min followed by 30 cycles of 94° C. for 1 min, 56° C. for 1 min and72° C. for 2 min. A final extension at 72° C. for 10 min completed thereactions. Approximately 1 to 5 μg of the PCR products were resolved on1% agarose gels and stained with ethidium bromide. PCR products of theappropriate size (300-350 base pairs) were inserted into the InvitrogenpCR2.1-TOPO-TA™ vector and transformed into IHV αF1 E. coli bacterialcells, according to the manufacturer's instructions. Colonies containingthe plasmid with insert were selected by overnight growth, at 37° C. onLuria-Bertani agar plates containing ampicillin (100 μg/ml) and X-gal(80 μg/ml). White (positive) colonies were incubated over night undershaking at 37° C. in 5 ml of Luria-Bertani broth containing ampicillin(100 μg/ml) and plasmids were isolated using QIAprep Spin Miniprep DNAisolation kits, according to the manufacturer's instructions (QIAGEN).EcoRI digestion of 1 to 5 μg of the resulting plasmids and resolution ofthe products by agarose gel electrophoresis, as described above,confirmed the presence of the appropriate size inserts. 1 to 5 putativepositive clones were sequenced with T7 (5′-TAA TAC GAC TCA CTA TAGGG-3′, SEQ ID NO: 19) and M13Rev (5′-CAG GAA ACA GCT ATG AC-3′, SEQ IDNO: 20) primers, using ABI Prism BigDye™ terminator cycle sequencingkits and ABI Prism 3100 Genetic Analyzer (Perkin Elmer). Consensussequences were generated using the DNASTAR software suite (DNASTARInc.). GenBank Accession Numbers are AY605265 to AY605273 for V_(H)sequences, and AY605274 to AY605282 for the V_(L).

Identification of Ig germline sequences and assignment of relevantregions—Consensus nucleotide sequences were compared against the Musmusculus immunoglobulin (Ig) set database using IMGT/V-Quest (Lefranc,2003; Hypertext Transfer Protocol://imgt (dot) cines (dot)fr/home.html). The sequences were concurrently compared against the Musmusculus Ig germline V-gene database using IgBlast (Aitschul et al.,1990 ; Hypertext Transfer Protocol://imgt (dot) cines (dot)fr/home.html://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot)gov/igblast/). This allowed the identification of the complementarydetermining region (CDR) and framework (FR) regions of the V_(H) andV_(L) sequences, and provided numbering to the inferred amino acidsequences according to Kabat et al. (1991). Similarly, the IgBlastresults allowed the identification of the most closely related murine Iggermline V-genes currently available in these databases. In all cases,the entire sequence, including those at the 5′ end of each sequenceimposed by the specific primers used in the original PCR amplification,were examined.

Results

Analysis of the V_(H) and V_(L) sequences of F8:33, F8:33-6-8-10 andMF1-16-11 murine mAbs is shown in FIGS. 18A-L.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications and GenBank Accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application or GenBank Accession numberwas specifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A fragment of a polypeptide encoded by a nucleic acid moleculecomprising the sequence of SEQ ID NO:1, the fragment having at least 6amino acids, wherein said nucleic acid molecule comprises the threeadditional nucleotides CAG in a region corresponding to the region ofnucleotides 908-910 in SEQ ID NO: 1, wherein the fragment retains theamino acid residues corresponding to residue 303 of SEQ ID NO:
 2. 2. Thepolypeptide fragment consisting of the amino acid sequence of SEQ ID NO:14, 15, 16, 17 or
 18. 3. A composition comprising the polypeptidefragment of claim 1.